https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Micah+ZileProteopedia - User contributions [en]2026-09-25T17:29:56ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332204Sandbox Reserved 18522025-04-28T20:02:55Z<p>Micah Zile: </p>
<hr />
<div>=Diels-Alderase=<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dienophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
The original enzyme was found using Rosetta parameters that were looking specifically for enzymes that contained two catalytic residues, specifically Tyr and Glu, and could coordinate two substrates highly specifically. Using the software, a large catalog of enzymes were screened and 207 scaffolds were screened for potential active site orientations that could accommodate the substrates. 84 of the 207 were selected for testing, 50 were found to be soluble, and only 2, DA_20_00 and DA_42_00 had any enzymatic activity. Preliminary results favored <scene name='10/1075254/Squidscaffold/2'>DA_20_00</scene> and thus this [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] scaffold was chosen as the base for further experiments.<ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref><br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed. The active site geometry also plays a large role in the binding of the substrates and how they react on a stereochemical level. By making small changes in the active site, changes can be made to the selectivity.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The original hypothesis was that including a steric group near to the top of the active site would increase the binding affinity of the enzyme and improve the reaction kinetics. It was experimentally shown that he hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref> <br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/> The authors did not publish comparisons to free reflux originally.<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332203Sandbox Reserved 18522025-04-28T20:02:28Z<p>Micah Zile: </p>
<hr />
<div>==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dienophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
The original enzyme was found using Rosetta parameters that were looking specifically for enzymes that contained two catalytic residues, specifically Tyr and Glu, and could coordinate two substrates highly specifically. Using the software, a large catalog of enzymes were screened and 207 scaffolds were screened for potential active site orientations that could accommodate the substrates. 84 of the 207 were selected for testing, 50 were found to be soluble, and only 2, DA_20_00 and DA_42_00 had any enzymatic activity. Preliminary results favored <scene name='10/1075254/Squidscaffold/2'>DA_20_00</scene> and thus this [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] scaffold was chosen as the base for further experiments.<ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref><br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed. The active site geometry also plays a large role in the binding of the substrates and how they react on a stereochemical level. By making small changes in the active site, changes can be made to the selectivity.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The original hypothesis was that including a steric group near to the top of the active site would increase the binding affinity of the enzyme and improve the reaction kinetics. It was experimentally shown that he hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref> <br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/> The authors did not publish comparisons to free reflux originally.<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332171Sandbox Reserved 18522025-04-28T18:57:51Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dienophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
The original enzyme was found using Rosetta parameters that were looking specifically for enzymes that contained two catalytic residues, specifically Tyr and Glu, and could coordinate two substrates highly specifically. Using the software, a large catalog of enzymes were screened and 207 scaffolds were screened for potential active site orientations that could accommodate the substrates. 84 of the 207 were selected for testing, 50 were found to be soluble, and only 2, DA_20_00 and DA_42_00 had any enzymatic activity. Preliminary results favored <scene name='10/1075254/Squidscaffold/2'>DA_20_00</scene> and thus this [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] scaffold was chosen as the base for further experiments.<ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref><br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed. The active site geometry also plays a large role in the binding of the substrates and how they react on a stereochemical level. By making small changes in the active site, changes can be made to the selectivity.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The original hypothesis was that including a steric group near to the top of the active site would increase the binding affinity of the enzyme and improve the reaction kinetics. It was experimentally shown that he hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref> <br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/> The authors did not publish comparisons to free reflux originally.<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332170Sandbox Reserved 18522025-04-28T18:55:31Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dienophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
The original enzyme was found using Rosetta parameters that were looking specifically for enzymes that contained two catalytic residues, specifically Tyr and Glu, and could coordinate two substrates highly specifically. Using the software, a large catalog of enzymes were screened and 207 scaffolds were screened for potential active site orientations that could accommodate the substrates. 84 of the 207 were selected for testing, 50 were found to be soluble, and only 2, DA_20_00 and DA_42_00 had any enzymatic activity. Preliminary results favored <scene name='10/1075254/Squidscaffold/2'>DA_20_00</scene> and thus this [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] scaffold was chosen as the base for further experiments.<ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref><br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed. The active site geometry also plays a large role in the binding of the substrates and how they react on a stereochemical level. By making small changes in the active site, changes can be made to the selectivity.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The original hypothesis was that including a steric group near to the top of the active site would increase the binding affinity of the enzyme and improve the reaction kinetics. It was experimentally shown that he hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref> <br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332166Sandbox Reserved 18522025-04-28T18:45:35Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed. The active site geometry also plays a large role in the binding of the substrates and how they react on a stereochemical level. By making small changes in the active site, changes can be made to the selectivity.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The original hypothesis was that including a steric group near to the top of the active site would increase the binding affinity of the enzyme and improve the reaction kinetics. It was experimentally shown that he hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref> <br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332160Sandbox Reserved 18522025-04-28T18:37:12Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332155Sandbox Reserved 18522025-04-28T18:26:40Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332152Sandbox Reserved 18522025-04-28T18:24:02Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the diene, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332144Sandbox Reserved 18522025-04-28T18:19:03Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the dienophile, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing the <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332142Sandbox Reserved 18522025-04-28T18:18:03Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the dienophile, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Tyr134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332139Sandbox Reserved 18522025-04-28T18:16:33Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the dienophile, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4332138Sandbox Reserved 18522025-04-28T18:16:20Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/3'><br />
<br />
==Introduction==<br />
The Diels-Alderase catalyzes the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction] [Fig. 1A] between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide [Fig. 1B] for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form [Fig.1C]. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
The Diels-Alderase was designed to connect a diene and dienophile to complete the Diels-Alder reaction. It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
[[Image:DielsAlderasecomboinfo Large.jpeg|540px|left|thumb|Figure 1. A) Example mechanism of a simple Diels-Alder reaction. B) Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide. C) Illustration of 3R, 4S endo stereoisomerism, which the Diels-Alderase is selective for.]]<br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand, 4-{[2-(phosphonooxy)ethyl]carbamoyl}benzyl [(1R,6S)-6-(dimethylcarbamoyl)cyclohex-2-en-1-yl]carbamate, in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile.<ref name="Siegel"/> It also contains a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
<br />
<br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile [Fig. 2]. Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene [Fig. 2]. These interactions help reduce the energetic gap between orbitals, allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|550px|left|thumb|Figure 3. Active site mechanism]]<br />
The key to the Diels-Alderase's success as a catalyst lies in its ability to lower the energy gap between reactants. To accomplish this, the two active site residues, Tyr134 and Glu208, use hydrogen bonding to assist the reaction in a variety of ways. <br />
<br />
First, it allows specific binding of the ligand in the active site, selecting for molecules with certain stereochemistry at and around the catalytic residues, specifically the [https://en.wikipedia.org/wiki/Carbamate carbamate] and [https://en.wikipedia.org/wiki/Carbonyl_group carbonyl] of the diene and dienophile, respectively. This promotes the reaction by stabilizing the molecules in close proximity to one another, also promoting the reaction's characteristic stereoselectivity.<ref name="Siegel"/> Second, the bonds affect the energetics of the molecules. By donating a hydrogen to the carbonyl of dienophile, Tyr134 helps to decrease the [https://en.wikipedia.org/wiki/Electron_density electron density] around the molecule, lowering the energy of the lowest unoccupied molecular orbital (LUMO).<ref name="Siegel"/> Conversely, by abstracting the hydrogen from the carbamate of the dienophile, Glu208 increases the electron density and thus the energy of the highest occupied molecular orbital (HOMO). By closing the gap between these orbitals, the enzyme lowers the [https://en.wikipedia.org/wiki/Activation_energy#:~:text=In%20the%20Arrhenius%20model%20of,mole%20(kcal%2Fmol). activation energy] required for the orbitals to react. Finally, these interactions help to stabilize the accumulated charges in the [https://en.wikipedia.org/wiki/Transition_state transition state.] By decreasing electron density in the dienophile, Tyr134 helps to stabilize the accumulated negative charge in the transition state. The Glu208, then, helps stabilize the accumulated positive charge by increasing the electron density of the diene. Calculations predict that this helps to stabilize the transition state by nearly 5 kcal/mol.<ref name="Siegel"/> All together, these interactions make it much easier for the reaction to proceed in a very stereoselective and favorable manner.<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/3'>serine</scene> to <scene name='10/1075254/A284/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:Introducing an <scene name='10/1075254/A285_scence/3'>alanine</scene> to <scene name='10/1075254/N285/5'>asparagine</scene> increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game [https://en.wikipedia.org/wiki/Foldit Foldit.]<ref name="Eiben"/> Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
The CE20 generation contains three highly conserved mutations found in many of the most catalytically efficient Diels-Alderase models being screened: Tyr43, Pro48, and Arg56 were mutated to <scene name='10/1075254/Ce_20_mutations/5'>Ile43, Lys48 and Ser56</scene>.<ref name="Preiswerk"/> Generally, these mutations each contributed to further tightening the binding pocket around the ligand and creating a more hydrophobic environment for enhanced binding.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity [Fig. 4].<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction.<ref name="Preiswerk"/> Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than the preferred values seen in any moderately-efficient natural enzymes catalyzing various reactions. This demonstrates the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution (about 10 substrate molecules/hour),<ref name="Preiswerk"/> the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in toluene for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331207Sandbox Reserved 18522025-04-24T02:38:33Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile (see Fig. 2). Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 3. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to assist in the reaction by closing the energy gap between the molecules. By donating a hydrogen, via Tyr134, the energy of the highest occupied molecular orbital (HOMO) is increased. Conversely, by abstracting a hydrogen, via Q208, the energy of the lowest unoccupied molecular orbital (LUMO) is decreased. As a result, the energy gap between the <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/3'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/1'>serine</scene> to <scene name='10/1075254/A285_scence/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/5'>Asp285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/5'>I43, L48 and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331206Sandbox Reserved 18522025-04-24T01:51:19Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile (see Fig. 2). Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 3. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to assist in the reaction by closing the energy gap between the molecules. By donating a hydrogen, via Tyr134, the energy of the highest occupied molecular orbital (HOMO) is increased. Conversely, by abstracting a hydrogen, via Q208, the energy of the lowest unoccupied molecular orbital (LUMO) is decreased. As a result, the energy gap between the <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/3'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/1'>serine</scene> to <scene name='10/1075254/A285_scence/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>Asp285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331205Sandbox Reserved 18522025-04-24T01:48:46Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile (see Fig. 2). Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>cap</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 3. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to assist in the reaction by closing the energy gap between the molecules. By donating a hydrogen, via Tyr134, the energy of the highest occupied molecular orbital (HOMO) is increased. Conversely, by abstracting a hydrogen, via Q208, the energy of the lowest unoccupied molecular orbital (LUMO) is decreased. As a result, the energy gap between the <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/3'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075254/S284/1'>serine</scene> to <scene name='10/1075254/A285_scence/1'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331204Sandbox Reserved 18522025-04-24T01:16:52Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile (see Fig. 2). Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>TextToBeDisplayed</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 3. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding site, and is more than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331203Sandbox Reserved 18522025-04-24T01:16:03Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075254/Y134_h_donation/3'>TextToBeDisplayed</scene> to the oxygen on the dienophile (see Fig. 2). Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075254/Alpha_helix_highlighted/1'>TextToBeDisplayed</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 3. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding site, and is more than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331202Sandbox Reserved 18522025-04-24T01:09:39Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 2. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075253/Y134_h_donation/3'>hydrogen bond donor</scene> to the oxygen on the dienophile (see Fig. 2). Q208 acts as a <scene name='10/1075254/208_bond_donor/3'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075252/Alpha_helix_highlighted/4'>motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 3. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding site, and is more than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure 4. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4331201Sandbox Reserved 18522025-04-24T00:47:03Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
Å <br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, using computational modeling and refinement through collaborative problem-solving from online users. The first generation Diels-Alderase was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]]<br />
The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. <br />
<br />
The binding pocket of 4o5t is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Tyr134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Glu208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the dieophile is in yellow and the diene is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/2'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/6'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the dienophile (see Fig. 3). Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075252/Alpha_helix_highlighted/4'>motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis], the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Tyr134 donates a hydrogen bond to the dieneophile, increasing its electron density and lowering its LUMO. Glu208 accepts a hydrogen bond from the diene, decreasing its electron density and lowering its HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models on various scaffolds, and researchers attempted to grow and purify those proteins within an ''E. coli'' host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding site, and is more than 3Å from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure X. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25°C, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]]<br />
<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''K<sub>m</sub>'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328714Sandbox Reserved 18522025-04-17T19:28:32Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using de novo enzyme design, which relies on computational modeling that is refined through programming collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|200px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>dienophile</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075252/Alpha_helix_highlighted/4'>motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding site entrance to the enzyme, and is more than 3 angstroms from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328711Sandbox Reserved 18522025-04-17T19:27:56Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using de novo enzyme design, which relies on computational modeling that is refined through programming collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.]<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>dienophile</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075252/Alpha_helix_highlighted/4'>motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding site entrance to the enzyme, and is more than 3 angstroms from the ligand in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. Choosing a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation increases the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. Introducing this mutation increases steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328692Sandbox Reserved 18522025-04-17T19:12:30Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>dienophile</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] <scene name='10/1075252/Alpha_helix_highlighted/4'>motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328691Sandbox Reserved 18522025-04-17T19:11:03Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the designed active site, <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> stabilize the transition state of the Diels-Alder reaction. The Tyr134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>dienophile</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the diene. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed.<br />
====Helix Cap====<br />
In the evolution process, a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the ''K<sub>m</sub>'' of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328687Sandbox Reserved 18522025-04-17T19:04:33Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328685Sandbox Reserved 18522025-04-17T19:03:59Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
<br />
[[Image:Diels-AlderaseSurfaces.png|300px|left|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328684Sandbox Reserved 18522025-04-17T19:03:32Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
<br />
[[Image:Diels-AlderaseSurfaces.png|300px|right|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328681Sandbox Reserved 18522025-04-17T19:02:47Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
<br />
[[Image:Diels-AlderasePocket.png|300px|right|thumb|Figure 3. Binding pocket and substrate. Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4328678Sandbox Reserved 18522025-04-17T19:00:52Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/2'><br />
<br />
==Introduction==<br />
[[Image:DielsAlderasesubstrates.png|300px|left|thumb|Figure 1. Diels-Alderase substrates]]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)]and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)]in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
<br />
The Diels-Alderase enzyme was built using de novo enzyme design, which relies on computational modeling that is refined through programming and collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/><br />
<br />
<br />
==General Structure==<br />
====Scaffold====<br />
After early Rosetta computational modelling, an ideal protein <scene name='10/1075254/Squidscaffold/1'>scaffold</scene> was found in the 6-bladed [https://en.wikipedia.org/wiki/Beta-propeller beta-propeller] of ''Loligo vulgalis,'' or the European Squid. <ref name="Siegel"/><ref name="Scharff">PMID:11435114</ref> The protein is relatively simple, with only one chain, one unit, 324 residues, and no extra ligands, metal ions, or small molecules bound. <br />
<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 3. Active site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075254/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue [https://proteopedia.org/wiki/index.php/Alpha_helix alpha-helix] motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction,” decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<ref name="Eiben">PMID:22267011</ref><br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure 4. Active site mechanism]]<br />
====HOMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction HOMO diene and the LUMO dienophile]. The goal of closing the energy gap allows the diene and dienophile to readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Bonding====<br />
Rather than using [https://en.wikipedia.org/wiki/Acid_catalysis acid-base catalysis]like many enzymes, the Diels-Alderase utilizes hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_00===<br />
During initial computer modelling, over one million potential Diels-Alderase active sites could be matched to potential protein scaffolds.<ref name="Siegel"/> Computer optimization narrowed this down to 84 potential models, and researchers attempted to grow and purify those proteins within an E. coli host. Of the 50 proteins that were successfully purified, only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS]screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening.<ref name="Siegel"/><ref name="Preiswerk"/> <br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
Glu 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 angstroms, increasing the strength of the interaction.<ref name="Siegel"/><br />
=====S284A=====<br />
:Ser 284 resides deep within the binding pocket of the enzyme. The group chose a <scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<ref name="Siegel"/><br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<ref name="Siegel"/><br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<ref name="Eiben"/><br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment.<ref name="Preiswerk"/><br />
<br />
==Kinetics==<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|360px|left|thumb|Figure X. Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 <ref name="Preiswerk">PMID:24847076</ref>]][[Image:Diels-AlderaseKineticgraph.jpg|350px|right|thumb|Figure X. Improvement of catalytic efficiency <ref name="Preiswerk"/>]]<br />
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (Km value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/><br />
<br />
<br />
<br />
<br />
<br />
<br />
==Applications==<br />
The CE20 model is the most efficient Diels-Alderase yet, surpassing many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<ref name="Preiswerk"/><br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the Diels-Alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <ref name="Preiswerk"/><br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=File:Diels-AlderaseSurfaces.png&diff=4328671File:Diels-AlderaseSurfaces.png2025-04-17T18:52:35Z<p>Micah Zile: Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The</p>
<hr />
<div>Shown is the binding pocket of the enzyme shown as surface, highlighting the electrostatics of the two catalytic residues, Tyr134 and Glu208. The ligand is color coded based on original structure: the diene is in yellow and the dienophile is in green. The reaction proceeds via attack of the C6 on the C5, shifting electron density to C2, which attacks C1.</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4326559Sandbox Reserved 18522025-04-16T00:19:04Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/1'><br />
<br />
==Introduction==<br />
[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
==General Structure==<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure #. Active site mechanism]]<br />
====HUMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the HOMO diene and the LUMO dienophile. The goal of closing the energy gap is so that in the diels-alder reaction, the diene and dienophile can readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Binding====<br />
Rather than using acid/base catalysis like many enzymes, the diels-alderase utilized hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Residue 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 Angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 Angstroms, increasing the strength of the interaction.<br />
=====S284A=====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose a<scene name='10/1075253/S284/2'>serine</scene> to <scene name='10/1075253/A285_scence/2'>alanine</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. <br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment. <br />
<br />
===Kinetics===<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|300px|left]]<br />
Catalytic efficiency of 4 key generations of Diels-Alderase enzyme. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 (cite 3)<br />
<br />
==Application==<br />
The CE20 model is up to 300 fold more efficient than the first generation model, making it the most efficient Diels-Alderase yet. It surpasses many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the diels-alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4326558Sandbox Reserved 18522025-04-16T00:17:12Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/1'><br />
<br />
==Introduction==<br />
[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
==General Structure==<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure #. Active site mechanism]]<br />
====HUMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the HOMO diene and the LUMO dienophile. The goal of closing the energy gap is so that in the diels-alder reaction, the diene and dienophile can readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Binding====<br />
Rather than using acid/base catalysis like many enzymes, the diels-alderase utilized hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Residue 162, a <scene name='10/1075254/Q162/2'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 Angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 Angstroms, increasing the strength of the interaction.<br />
=====S284A=====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/2'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. <br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment. <br />
<br />
===Kinetics===<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|300px|left]]<br />
Catalytic efficiency of 4 key generations of Diels-Alderase enzyme. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 (cite 3)<br />
<br />
==Application==<br />
The CE20 model is up to 300 fold more efficient than the first generation model, making it the most efficient Diels-Alderase yet. It surpasses many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the diels-alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4326557Sandbox Reserved 18522025-04-16T00:14:43Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/1'><br />
<br />
==Introduction==<br />
[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
==General Structure==<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure #. Active site mechanism]]<br />
====HUMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the HOMO diene and the LUMO dienophile. The goal of closing the energy gap is so that in the diels-alder reaction, the diene and dienophile can readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Binding====<br />
Rather than using acid/base catalysis like many enzymes, the diels-alderase utilized hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Residue 162, a <scene name='10/1075254/Q162/1'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is outside 3 Angstroms in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose to mutate this Q to an <scene name='10/1075254/Q_to_r/1'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5 Angstroms, increasing the strength of the interaction.<br />
=====S284A=====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/2'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. <br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment. <br />
<br />
===Kinetics===<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|300px|left]]<br />
Catalytic efficiency of 4 key generations of Diels-Alderase enzyme. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 (cite 3)<br />
<br />
==Application==<br />
The CE20 model is up to 300 fold more efficient than the first generation model, making it the most efficient Diels-Alderase yet. It surpasses many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the diels-alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4326556Sandbox Reserved 18522025-04-16T00:10:37Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/1'><br />
<br />
==Introduction==<br />
[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
==General Structure==<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure #. Active site mechanism]]<br />
====HUMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the HOMO diene and the LUMO dienophile. The goal of closing the energy gap is so that in the diels-alder reaction, the diene and dienophile can readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Binding====<br />
Rather than using acid/base catalysis like many enzymes, the diels-alderase utilized hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Residue 162, a <scene name='10/1075254/Q162/1'>glutamine</scene>, resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to <scene name='10/1075254/Q_to_r/1'>R mutation</scene>, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
=====S284A=====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/2'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. <br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment. <br />
<br />
===Kinetics===<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|300px|left]]<br />
Catalytic efficiency of 4 key generations of Diels-Alderase enzyme. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 (cite 3)<br />
<br />
==Application==<br />
The CE20 model is up to 300 fold more efficient than the first generation model, making it the most efficient Diels-Alderase yet. It surpasses many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the diels-alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4326553Sandbox Reserved 18522025-04-15T23:48:49Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/1'><br />
<br />
==Introduction==<br />
[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
==General Structure==<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure #. Active site mechanism]]<br />
====HUMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the HOMO diene and the LUMO dienophile. The goal of closing the energy gap is so that in the diels-alder reaction, the diene and dienophile can readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Binding====<br />
Rather than using acid/base catalysis like many enzymes, the diels-alderase utilized hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to <scene name='10/1075254/Q_to_r/1'>R mutation</scene>, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
=====S284A=====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/2'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
=====A285N=====<br />
:<scene name='10/1075254/N285/3'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. <br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment. <br />
<br />
===Kinetics===<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|300px|left]]<br />
Catalytic efficiency of 4 key generations of Diels-Alderase enzyme. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 (cite 3)<br />
<br />
==Application==<br />
The CE20 model is up to 300 fold more efficient than the first generation model, making it the most efficient Diels-Alderase yet. It surpasses many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the diels-alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1852&diff=4326552Sandbox Reserved 18522025-04-15T23:44:01Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Diels-Alderase ==<br />
<StructureSection load='4o5t' size='340' side='right' caption='Diels-Alderase 4o5t' scene='10/1075254/Front_Page/1'><br />
<br />
==Introduction==<br />
[https://www.pnas.org/doi/full/10.1073/pnas.1401073111 Impact of Scaffold Rigidity]<br />
The Diels-Alderase protein aims to create optimal reacting conditions between the diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital (LUMO) and the diene’s highest occupied molecular orbital (HOMO) in the transition state.<ref name="Siegel">PMID:20647463</ref> The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate (diene) and N,N- dimethylacrylamide (dienophile). The binding site contains an H-bond donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and an H-bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene. Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. <br />
==General Structure==<br />
[[Image:BindingPocket.png|300px|right|thumb|Figure 1. Active Site]]<br />
====Active Site====<br />
In the active state, there are <scene name='10/1075254/Active_site/3'>two catalytic residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. The Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/6'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
====Helix Cap====<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
<br />
== Mechanism ==<br />
[[Image:Resizedmechanism.png|500px|left|thumb|Figure #. Active site mechanism]]<br />
====HUMO and LUMO====<br />
The two active site residues, Y134 and Q208, use hydrogen bonding to close the energy gap between the HOMO diene and the LUMO dienophile. The goal of closing the energy gap is so that in the diels-alder reaction, the diene and dienophile can readily switch roles for the mechanism to progress and complete the formation of the product. Due to the conserved nature of this mechanism, the diels-alderase is stereoselective for the 3R, 4S endo product. <br />
====Hydrogen Binding====<br />
Rather than using acid/base catalysis like many enzymes, the diels-alderase utilized hydrogen bonding to alter the HOMO and LUMO energies of the diene and dienophile. Y134 donates a hydrogen bond to the dieneophile, increasing the electron density and lowering the LUMO. Q208 accepts a hydrogen bond from the diene, decreasing the electron density and lowering the HOMO. <br />
<br />
<br />
<br />
<br />
<br />
==Development and Evolution==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
=====Q162R=====<br />
:Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to <scene name='10/1075254/Q_to_r/1'>R mutation</scene>, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
=====S284A=====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/2'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
=====A285N=====<br />
:<scene name='10/1075254/N285/2'>N285</scene>, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. <br />
===CE6===<br />
The DA_20_10 model of the Diels Alderase was further enhanced by players of the online game "Foldit." Building on preliminary early data, players were asked to optimize various helical structures that would surround and support the ligand. After over 100,000 designs were tested, the top-scoring CE6 model was finalized, containing as <scene name='10/1075252/Alpha_helix_highlighted/4'>alpha helix cap</scene> that favorably constrains ligand orientation. This "cap" consists of two helices--helix one spans from residues 36-44, and helix two spans from residues 48-56.<br />
===CE20===<br />
In this generation, it was found that the most catalytically efficient models had mutated T34, P48, and R56 to <scene name='10/1075254/Ce_20_mutations/4'>I43,L48, and S56</scene>. These mutations further tightened the binding pocket and create a more hydrophobic environment. <br />
<br />
===Kinetics===<br />
[[Image:Diels_Alderase_kinetic_table_Large.jpeg|300px|left]]<br />
Catalytic efficiency of 4 key generations of Diels-Alderase enzyme. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4 (cite 3)<br />
<br />
==Application==<br />
The CE20 model is up to 300 fold more efficient than the first generation model, making it the most efficient Diels-Alderase yet. It surpasses many other biological (antibody) and artificial (ribozyme, metalloenzyme) attempts at catalyzing the Diels-Alder reaction. Even then, the CE20 model has a catalytic efficiency value at least 4 orders of magnitude lower than those seen in other moderately-efficient natural enzymes, demonstrating the innate slowness of the Diels-Alder reaction.<br />
<br />
Though the rate of product formation using this enzyme is not significantly different from that found when reactants reflux free in solution, the diels-alderase shows a vast improvement in product stereoselectivity. When refluxed in a room temperature aqueous solution containing the necessary substrates, the enzyme catalyzed an over 90% conversion rate, producing only the 3R,4S endo cyclohexane product isomer. By comparison, refluxing the substrates free in solution for a similar duration of time yields a racemic (66:34) mixture of endo and exo products. It is primarily for these stereoselective benefits that this enzyme is valuable for synthetic purposes. <br />
<br />
Future improvement of the Diels-Alderase will likely revolve around the improvement of catalytic efficiency, further constriction of the active site, and selective production of varying stereoisomers.<br />
<br />
== References ==<br />
<references/><br />
<br />
==Student Contributors==<br />
Taylor Donahue, Kate Thuma, Micah Zile</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4326550Sandbox Reserved 18512025-04-15T23:41:59Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/4'<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/5'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/2'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4326549Sandbox Reserved 18512025-04-15T23:40:09Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/4'<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/5'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to <scene name='10/1075253/A285_scence/1'>A</scene> mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4326545Sandbox Reserved 18512025-04-15T23:20:23Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/4'<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Y134_h_donation/1'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/208_bond_donor/1'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/5'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose an <scene name='10/1075253/S284/2'>S</scene> to A mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also changing any steric characteristics in the region ''unintentionally'' near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325760Sandbox Reserved 18512025-04-10T19:55:28Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/4'<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/5'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=File:N285PDB.pdb&diff=4325744File:N285PDB.pdb2025-04-10T19:23:17Z<p>Micah Zile: N285 mutation for IS10</p>
<hr />
<div>N285 mutation for IS10</div>Micah Zilehttps://proteopedia.org/index.php?title=File:A285PDB.pdb&diff=4325743File:A285PDB.pdb2025-04-10T19:23:03Z<p>Micah Zile: A285 mutation for IS10</p>
<hr />
<div>A285 mutation for IS10</div>Micah Zilehttps://proteopedia.org/index.php?title=File:S284PDB.pdb&diff=4325742File:S284PDB.pdb2025-04-10T19:22:45Z<p>Micah Zile: S284 original PDB for IS10</p>
<hr />
<div>S284 original PDB for IS10</div>Micah Zilehttps://proteopedia.org/index.php?title=File:R162PDB.pdb&diff=4325741File:R162PDB.pdb2025-04-10T19:22:22Z<p>Micah Zile: R162 mutation for IS10</p>
<hr />
<div>R162 mutation for IS10</div>Micah Zilehttps://proteopedia.org/index.php?title=File:Q162PDB.pdb&diff=4325740File:Q162PDB.pdb2025-04-10T19:21:35Z<p>Micah Zile: Original Q amino acid for IS10</p>
<hr />
<div>Original Q amino acid for IS10</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325738Sandbox Reserved 18512025-04-10T19:19:59Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/3'<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/5'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325722Sandbox Reserved 18512025-04-10T19:06:40Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/2<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/5'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325717Sandbox Reserved 18512025-04-10T18:59:02Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/2<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/2'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325713Sandbox Reserved 18512025-04-10T18:55:56Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/2<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue <scene name='10/1075253/Helix/1'>a-helix motif</scene> to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325708Sandbox Reserved 18512025-04-10T18:50:38Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/2<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme.<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zilehttps://proteopedia.org/index.php?title=Sandbox_Reserved_1851&diff=4325680Sandbox Reserved 18512025-04-10T18:19:16Z<p>Micah Zile: </p>
<hr />
<div>{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --><br />
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design==<br />
<Structure load='4o5t' size='350' frame='true' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/2<br />
' /><br />
==Introduction==<br />
[https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder Reaction]<br />
==General Structure==<br />
===Active Site===<br />
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO.<br />
===Helix Addition===<br />
<br />
==Mechanism==<br />
===Uncatalyzed Reaction===<br />
===Orbital Stabilization===<br />
<br />
==Structural Details==<br />
===Active Site===<br />
[[Image:N285toY134Mutant_(1).png|400 px|left|thumb|Figure 1. The coolest image of this protein EVAH!!!]]<br />
<scene name='10/1075253/Active_site_residues/2'>Active Site Residues</scene><br />
==Development & Evolution ==<br />
===DA_20_10===<br />
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues.<br />
====Q162R====<br />
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction.[[Image:Q162R.jpeg]]<br />
====S284A====<br />
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues.[[Image:S284A.jpeg]]<br />
====A285N====<br />
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand.[[Image:A285N.jpeg]]<br />
<br />
== Relevance ==<br />
===Chemical Applications===<br />
===Improvements===<br />
<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/><br />
<br />
==Student Collaborators==<br />
Micah Zile<br />
Kate Thuma<br />
Taylor Donahue</div>Micah Zile