Ketosteroid Isomerase: Difference between revisions
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====Beta-Turns and Loops==== | ====Beta-Turns and Loops==== | ||
β-strand 1 is connected to β-strand 2 via a four residue linkage (Pro39 to Ser42) with Pro39 being in a cis-conformation. Two examples of type-II β-turns can also be observed in KSI-between β-stands 3 & 4 (Ala75-Asn76→see figure) and β-strands 4 & 5 (Gln89-Gly90). A loop structure (Asn105-Val107) also connects β-strands 5 and 6. | |||
[[Image: | |||
===Dimer Interface=== | |||
The biologically active unit of KSI is a 2-fold symmetric dimer in which the two chains are packed together via hydrophobic and electrostatic interactions between the "back faces" of the β-sheets. The curved β-sheets on each of the monomers expose convex faces to each other, forming well-defined interactions. In their NMR structure of KSI, Massiah et al. identified interchain hydrophobic interactions (A), as well, a number of polar residues (B) located within the dimer interface. Although they were unable to resolve interchain hydrogen-bonding interactions, they were able to resolve an intrachain hydrogen bond between His100 and Glu77 (C). The apparent pKa of His100 is 4.3, and the authors speculate that this unusually low pKa may be the result of electrostatic effects from nearby cationic residues (Arg102 and Arg113). | |||
[[Image:Massiah.png|center]] | |||
In their X-ray crystal structure of KSI, Kim et al. identified bound water molecules within the dimer interface which may mediate hydrogen-bonds between Thr68, Arg72, and Asp96. The water mediated hydrogen bond between Thr68 and Arg72 is illustrated below. Kim et al. also identified interchain hydrogen bonds between the backbone carbonyl oxygens of Val71, Ala73, and Val97. | |||
[[Image:Kim1.png|left]] [[Image:Kim2.png|center]] | |||
===Hydrophobic Active Site=== | |||
[[Image:phobia.png|left]] | |||
KSI's active site is located within a hydrophobic cavity formed helices B and C crossing over the "front face" of the β-sheet that is approximately 8.5 by 9.5 Å at its opening and is 16 Å deep.<ref name="Wu" /> The cavity is lined with <scene name='User:Laura_M._Haynes/Sandbox_1/Beta-phobics/1'>hydrophobic "front facing" residues</scene> from the β-sheet(Val36, Pro39, Leu63, Val65, Leu67, Val71, Phe80, Phe82, Val84, Val95, Pro97, Phe101, Ala114, and Phe116). <scene name='User:Laura_M._Haynes/Sandbox_1/Helix-phobic/1'>Hydrophobic residues on the other side of the cavity</scene> contributed from the α-helices include: Val11, Tyr14, Val15, Leu18, Phe54, and Tyr55.<ref name="Wu" /> | |||
Although the <scene name='User:Laura_M._Haynes/Sandbox_1/Active_site_1isk/1'>active site</scene> of KSI is notably hydrophobic, it contains several hydrophilic residues believed to be important to the enzymatic function of the protein. The hydrophobic active site of KSI contains an aspartate residue at position 99 and a tyrosine residue at position 14 (according to the numbering for the ''Commamonas tetosteroni'' protein, which will be used throughout) that are capable of forming hydrogen bonds with the 3-position carbonyl of the steroid and form an active site oxyanion hole.<ref name="Pollack" />,<ref name="Sigala2008">PMID:18808119 </ref> Additionally, the active site contains an aspartate residue at position 38 that is participates in the catalytic activity of KSI.<ref name="Pollack" /> [[Image:cis.png|right]] | |||
Upon substrate binding the the three α-helices become more tightly packed with the "front face" of the β-sheet. This in turn allows Tyr14 to approach Asp99 and the substrate. | |||
[[Image:morph.mov|center]] | |||
===Cis-Peptide Bond=== | |||
Pro39 of KSI participates in a cis-peptide linkage with Asp38 in forming a <scene name='User:Laura_M._Haynes/Sandbox_1/Cis_loop/1'>four-residue linkage</scene>between stands 1 and 2 of the 6 stranded beta-sheet.<ref name="Wu" /> The cis-peptide linkage serves to correctly position the key catalytic residue Asp38 within the active site. Mutating residue to Gly or Ala results in the improper positioning of Asp38 within the active site leading to corresponding 2-fold decreases in enzyme's catalytic efficiency.<ref name="NAM">PMID:12852789 </ref> The cis-peptide bond also helps to stablize and increase the rigidity of the four-residue linkage between β-strands 1 and 2.<ref name="NAM" /> | |||
==Structural Classifications== | |||
===CATH=== | |||
KSI is classified using the CATH classification system as follows: | |||
# Class - Alpha Beta | |||
# Architecture - Roll | |||
# Topology - Nuclear Transport Factor 2 | |||
===SCOP=== | |||
KSI is classified using the SCOP classification system as follows: | |||
# Class - Alpha and Beta (α+β) | |||
# Fold - Cystatin-like | |||
# Superfamily - NTF2-like | |||
# Family - Ketosteroid Isomserase-like | |||
# Domian - Δ5-3-ketosteroid isomerase | |||
==Enzyme Mechanism== | |||
<applet load='1QJG' size='300' frame='true' align='left' caption='A monomer of ketosteroid isomerase is shown in complex with the transition state analog [http://en.wikipedia.org/wiki/Equilenin equinelin] (PBD Structure ID [[1qjg|1QJG]]).' scene='User:Laura_M._Haynes/Sandbox_1/1qjg/1'/> | |||
===General Mechanism=== | |||
The general mechanism of the proposed reaction of ketosteroid isomerase involves the breaking of a C-H bond adjacent to a carbonyl. This is typically regarded as a difficult reaction due to instability of the intermediate; however it is observed in a number of enzyme-mediated biological reactions.<ref name="Pollack" /> In line with other biological reactions, the mechanism of KSI involves the abstraction the β-hyrdogen from the 4-position carbon resulting in the formation of an enol intermediate, which is followed by reketonization.<ref name="Pollack" />,<ref name="Ha" /> Structural and kinetic studies suggest that Asp<sup>38</sup> (numbering is that of the TI varient of KSI) serves as a general base in this reaction as shown below and abstracts the β-proton from C4 with the ''syn'' orbitals of its carboxylate group.<ref name="Wu" /> Note the formation of the unstable enolate intermediate. | |||
[[Image:General_base.jpg|center]] | |||
Tyr<sup>14</sup> and Asp<sup>99</sup> are believed to participate in hydrogen bonding to the O-3 carbonyl of the substrate steroid and stabilize reaction intermediates. Tyr<sup>14</sup> is also believed to participate in a low barrier hydrogen bond with the 3-position oxygen of the steroid, thereby facilitating the abstraction of the β-hydrogen at the 4-position. There are two proposed models of this hydrogen bonding. In <scene name='User:Laura_M._Haynes/Sandbox_1/Model_1_1qjg/1'>Model 1</scene>, Tyr<sup>14</sup> and Asp<sup>99</sup> are both bound to the 3-position oxygen, whereas, in <scene name='User:Laura_M._Haynes/Sandbox_1/Model_2_1qjg/1'>Model 2</scene>, they form a hydrogen bonding network. These are <scene name='User:Laura_M._Haynes/Sandbox_1/1qjg/1'>shown here</scene> as a single monomer in complex with the intermediate analog [http://en.wikipedia.org/wiki/Equilenin equilenin].<ref name="Cho2">PMID:10551849</ref> | |||
Model 1 has become the generally accepted scheme through both crystallographic and mutanagenic/kinetic approaches. Mutation of Asp<sup>99</sup> to alanine and Tyr<sup>14</sup> to phenylalanine resulted in deleterious effects on kinetic parameters, which were additive in nature suggesting that Tyr<sup>14</sup> and Asp<sup>99</sup> participate equally in hydrogen binding with the oxygen atom. Additionally, the crystal structure of TI analog from ''Pseudomonas putida'' supports this conclusion given the <scene name='User:Laura_M._Haynes/Sandbox_1/Model_1_1qjg/1'>apparent hydrogen bonding distances</scene>.<ref name="Pollack" /> The general mechanism in the active site of KSI is outlined below. | |||
[[Image:H_bond.jpg|center]] | |||
===Low Barrier Hydrogen Bond=== | |||
Ketosteroid isomerase has become a ''test'' enzyme in the debate over the existence of [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bonds] (LBHBs) in accordance with the hydrogen bonding <scene name='User:Laura_M._Haynes/Sandbox_1/Model_1_1qjg/1'>Model 1</scene> discussed above. [http://en.wikipedia.org/wiki/NMR Nuclear magnetic resonance (NMR)] studies of KSI in complex with transition state analogs have revealed the presence of a highly shielded proton characteristic of the formation of a LBHB between Tyr<sup>14</sup> and the O-3 atom of the analogs and suggestive of the formation of a bridging hydrogen with short bonds.<ref name="Ha" />,<ref name="Zhao1996">PMID:8710850 </ref>,<ref name="Cleland1998" /> Additionally, NMR [http://en.wikipedia.org/wiki/Equilibrium_fractionation fractionation] studies with deuterium substitution are strongly suggestive of the high strength of this bond as deuterium is retained preferentially in this position.<ref name="Zhao1996" /> The energy of this bond along with that of the normal hydrogen bond from Asp<sup>99</sup> most likely contribute to the energy needed to support proton abstraction from the C-4 position.<ref name="Ha" /> | |||
===LBHB Controversy and Catalytic Efficiency from Preorganized State=== | |||
In their 2002 computational study Feierberg and Aquist propose that KSI is not the site of LBHB mediated catalysis<ref name="Feierberg2002">PMID:8710850 </ref>, a viewpoint that is shared among a subset of enzymologists.<ref name="Kraut2010">PMID:20080683 </ref> Hershlag and coworkers are proponents of the hydrogen bonding mechanism described in <scene name='User:Laura_M._Haynes/Sandbox_1/Model_1_1qjg/1'>Model 1</scene>;<ref name="Kraut2010" />,<ref name="Sigala2009">PMID:19469568 </ref> however, he suggests that hydrogen bonding is important within the active site of KSI albeit in the form of multiple weaker forces.<ref name="Kraut2006" /> Hydrogen bonds and other electrostatic contributions are contended to allow for a degree of preorientation of the active site for transition state stabilization that helps to counter act the effects of desolvation and reorientation of dipoles within the enzyme active site to support the oxyanion hole, which helps to avoid "sluggish" behavior associated with such rearrangement.<ref name="Pollack" /> This type of hydrogen bonding network also helps to disperse negative charge enabling stabilization of the transition state because in the hydrophobic environment of the active site large charges are abhorred.<ref name="Kraut2006" /> | |||
===Implications of Hydrophobic Active Site=== | |||
In combination with the above mentioned hydrogen bond interactions between the substrate and the active site, the highly hydrophobic active site also serves to stabilize the catalytic efficiency of KSI because it is in a preorganized state compared to the of the reaction carried out in solvent where [http://en.wikipedia.org/wiki/Acetate acetate anion] is used to mimic the functionality of Asp<sup>38</sup> and has a similar [http://en.wikipedia.org/wiki/PKa pKa]of 4.6 as suggested above. The consequence of this hydrophic active site is that Asp<sup>38</sup> has no neighbors capable of hydrogen bonding interactions and as such one may suspect its pKa to raised in this environment. Maintenance of this ''normal'' pKa is important because protonation of Asp<sup>38</sup> would no longer permit it to carry out hydrogen abstraction. To prevent divergence of Asp<sup>38</sup> from its ''normal'' pKa during initial substrate binding, it is believe hydrogen bonded to two or three water molecules, as suggested by computer models, which are excluded from the active site later in the enzymatic reaction.<ref name="Pollack" /> | |||
==Related Proteins== | |||
===Functionally Related Proteins=== | |||
Ketosteroid isomerase is one of a large number of enzymes that catalyze the cleavage of a C-H bond. Among these are [http://en.wikipedia.org/wiki/Mandelate_racemase mandelate racemace], [[Triosephosphate Isomerase|triosephosphate isomerase]], [http://en.wikipedia.org/wiki/Citrate_synthase citrate synthase], and [http://en.wikipedia.org/wiki/4-Oxalocrotonate_tautomerase 4-oxalocrotonate tautomerase].<ref name="Pollack" /> | |||
===Structural Homologs=== | |||
As noted above, [http://en.wikipedia.org/wiki/Scytalone_dehydratase scytalone dehydratase], [http://en.wikipedia.org/wiki/NUTF2 nuclear transport factor 2 (NTF2)], and [http://en.wikipedia.org/wiki/Naphthalene_1,2-dioxygenase naphthalene 1,2-dioxygenase] share similar structural motifs to ketosteroid isomerase, which facilitate binding of hydrophobic substrates.<ref name="Ha" />,<ref name="Murzin" /> It has also been postulated that bile acid 7α-dehydratase is also a member of this family of protein and that it shares functional similarity with KSI.<ref name="Murzin" /> In a recent paper, Cherney ''et al''<ref name="Cherney">PMID:18589008 </ref>. identified ''Myobacterium tuberculosis'' Rv0760c as a structural homolog of KSI. Although these proteins share similar structural motifs there carry out a diverse array of functions.<ref name="Cherney" /> | |||
==Available Structures== | |||
*[[1isk]]-First solution phase structure NMR structure of KSI from ''Comamonas testosteroni'' | |||
*[[1opy]] | |||
*[[1buq]] | |||
*[[1qjg]]-KSI from ''Pseudomoas testosteroni'' in complex with equilenin | |||
*[[1c7h]] | |||
*[[1e3r]] | |||
*[[1e3v]] | |||
*[[1e97]] | |||
*[[1ea2]] | |||
*[[1k41]] | |||
*[[1gs3]] | |||
*[[1ogx]] | |||
*[[1oh0]]-KSI from ''Pseudomonas putida'' in complex with equilenin | |||
*[[1cqs]] | |||
*[[1vzz]] | |||
*[[1w01]] | |||
*[[1w02]] | |||
*[[1oho]]-KSI Y16F/D40N mutant from ''Pseudomonas putida'' in complex with equilenin | |||
*[[1w6y]] | |||
*[[1w0o]] | |||
*[[2pzv]] | |||
*[[2inx]] | |||
*[[3cpo]] | |||
*[[3ex9]] | |||
*[[3fzw]] | |||
*[[3ipt]] | |||
*[[3m8c]] | |||
==References== | |||
<references /> | |||
