Sandbox Reserved 1846: Difference between revisions
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=== Phe243 === | === Phe243 === | ||
<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand, <scene name='10/1075247/Original_15_mutation_structure/6'>Reference Phe243</scene>. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The larger tryptophan at position 243 stabilizes the ligand through stronger hydrophobic and π–π interactions, pulling it closer to the catalytic site despite its size. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. π-stacking]. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme.<ref name="Tournier"/> | |||
<scene name='10/1075247/F243_original/1'>Phe243</scene> is located 3.6 Å from the ligand, <scene name='10/1075247/Original_15_mutation_structure/6'>Reference Phe243</scene>. Two mutations at this position, <scene name='10/1075247/F243i/1'>F243I</scene> and F243W, increase the catalytic activity of the enzyme. The F243I mutation inserts the smaller isoleucine whose side chain allows the ligand to sit closer. This reduces the ligand distance to 3.0 Å, improving substrate binding. The F243W mutation inserts the bulkier, nitrogen-containing aromatic aide chain. Trp brings the ligand slightly closer at 3.2 Å and introduces potential for new interactions, such as hydrogen bonding or [https://en.wikipedia.org/wiki/Pi-stacking#:~:text=In%20chemistry%2C%20pi%20stacking%20(also,interaction%22)%20is%20electrostatically%20repulsive. π-stacking]. Both mutations result in improved catalytic performance. The F243I mutant shows a 27.5% increase in activity, while the F243W mutant shows a 17.5% increase, compared to the wild-type enzyme.<ref name="Tournier"/> | |||
=== Tyr 127 === | === Tyr 127 === | ||
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=== Ser 283 & Asp 238 === | === Ser 283 & Asp 238 === | ||
Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>Reference Ser283 and Asp238</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/4ebo_disulfide_bond_skyblue/2'>S283C and D238C mutation</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function. | Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>Reference Ser283 and Asp238</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/4ebo_disulfide_bond_skyblue/2'>S283C and D238C mutation</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function. | ||
== Group Mutations == | == Group Mutations == | ||
These mutations were combined to create multi-mutant LCC variants with improved activity and thermostability. The two most successful variants were: | These mutations were combined to create multi-mutant LCC variants with improved activity and thermostability. The two most successful variants were: | ||
ICCG | The ICCG variant (F243I/D238C/S283C/Y127G) showed a 1.22-fold higher specific activity than wild-type LCC, with a melting temperature increase of +9.3°C. It achieved 90% PET depolymerization in 9.3 hours at 72°C. | ||
WCCG | The WCCG variant (F243W/D238C/S283C/Y127G) had specific activity similar to or slightly lower than ICCG, but showed even greater thermostability, with a melting temperature increase of +10.1°C. It reached 90% PET depolymerization in 10.5 hours at 72°C. | ||
Other stabilizing mutations, such as T96M, N246D, and N246M, were also tested but are not included in this page's protein model. These were excluded because they were not part of the top-performing mutant (ICCG), and therefore omitted for clarity.<ref name="Tournier"/> | Other stabilizing mutations, such as T96M, N246D, and N246M, were also tested but are not included in this page's protein model. These were excluded because they were not part of the top-performing mutant (ICCG), and therefore omitted for clarity.<ref name="Tournier"/> | ||
