Ketosteroid Isomerase

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This reaction is essential in the biosynthesis of steroids in mammals where KSI is a membrane-bound complex.<ref name="Ha">PMID:11751047</ref> In bacteria, however, KSI exists as a soluble protein is involves in catabolism of steroids.<ref name="Ha" /> It was first isolated in and has been extensively studied in [http://en.wikipedia.org/wiki/Comamonas_testosteroni ''Commamonas tetosteroni''] (TI), a bacteria that is capable of growth with testosterone as its sole carbon source.<ref name="Stanier1966">PMID:11751047</ref> Structural and kinetic studies of this and its homolog from [http://en.wikipedia.org/wiki/Pseudomonas_putida ''Pseudomonas putida''] with which it shares 34% sequence and near identical structural homology.<ref name="Pollack" />,<ref name="Ha" /> It is one of the most efficient known enzymes with an essentially diffusion limited rate of catalysis.<ref name="Talalay" />,<ref name="Wu">PMID:9103200 </ref> It is capable of increasing the catalytic rate by eleven orders of magnitude.<ref name="Murzin">PMID:9666335 </ref>
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This reaction is essential in the biosynthesis of steroids in mammals where KSI is a membrane-bound complex.<ref name="Ha">PMID:11751047</ref> In bacteria, however, KSI exists as a soluble protein is involves in catabolism of steroids.<ref name="Ha" /> It was first isolated in and has been extensively studied in [http://en.wikipedia.org/wiki/Comamonas_testosteroni ''Commamonas tetosteroni''] (TI), a bacteria that is capable of growth with testosterone as its sole carbon source.<ref name="Stanier1966">PMID:11751047</ref> Structural and kinetic studies of this and its homolog from [http://en.wikipedia.org/wiki/Pseudomonas_putida ''Pseudomonas putida''] with which it shares 34% sequence and near identical structural homology.<ref name="Pollack" />,<ref name="Ha" /> It is one of the most efficient known enzymes with an essentially diffusion limited rate of catalysis.<ref name="Talalay" />,<ref name="Wu">PMID:9103200 </ref> It is capable of increasing the catalytic rate by eleven orders of magnitude.<ref name="Murzin">PMID:9666335 </ref> Its high catalytic efficiency and unique active site geometry have made it fertile ground for examining the validity of the [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond low barrier hydrogen bond] hypothesis<ref name="Cleland1998">PMID:9748211 </ref> and electrostatic preorganization.<ref name="Kraut2006">PMID:16602823 </ref>.
==Structure==
==Structure==
Ketosteroid isomerase exits as a 28 kDa homodimeric protein, in which the two dimers related to each other via hydrophobic and electrostatic interactions.<ref name="Wu" /> Each monomer consists of a curved <scene name='User:Laura_M._Haynes/Sandbox_1/Beta_sheets/1'>beta-sheets</scene> and three <scene name='User:Laura_M._Haynes/Sandbox_1/Alpha_helices/1'>alpha-helices</scene>. These secondary structures define a conical closed barrel geometry, with one open and one closed end, and create a deep pocket in which the active site resides.<ref name="Ha" />,<ref name="Cho">PMID:9622484 </ref> This unique geometry is shared by several other proteins (scytalone dehydratase, nuclear transport factor 2, and naphthalene 1,2-dioxygenase), however, these molecules do not share functional or sequence homology. It is speculated that this unique protein structure may enable better binding of hydrophobic substrates such as steroids.<ref name="Ha" />
Ketosteroid isomerase exits as a 28 kDa homodimeric protein, in which the two dimers related to each other via hydrophobic and electrostatic interactions.<ref name="Wu" /> Each monomer consists of a curved <scene name='User:Laura_M._Haynes/Sandbox_1/Beta_sheets/1'>beta-sheets</scene> and three <scene name='User:Laura_M._Haynes/Sandbox_1/Alpha_helices/1'>alpha-helices</scene>. These secondary structures define a conical closed barrel geometry, with one open and one closed end, and create a deep pocket in which the active site resides.<ref name="Ha" />,<ref name="Cho">PMID:9622484 </ref> This unique geometry is shared by several other proteins (scytalone dehydratase, nuclear transport factor 2, and naphthalene 1,2-dioxygenase), however, these molecules do not share functional or sequence homology. It is speculated that this unique protein structure may enable better binding of hydrophobic substrates such as steroids.<ref name="Ha" />
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Although the <scene name='User:Laura_M._Haynes/Sandbox_1/Active_site_1isk/1'>active site</scene> of KSI is notably <scene name='User:Laura_M._Haynes/Sandbox_1/Hydrophobic/1'>hydrophobic (residues shown in gray) </scene>, 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 binding the 3-position carbonyl of the steroid. Additionally, the active site contains an aspartate residue at position 38 that is participates in the catalytic activity of KSI.<ref name="Pollack" />
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Although the <scene name='User:Laura_M._Haynes/Sandbox_1/Active_site_1isk/1'>active site</scene> of KSI is notably <scene name='User:Laura_M._Haynes/Sandbox_1/Hydrophobic/1'>hydrophobic (residues shown in gray) </scene>, 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" />
==Enzyme Mechanism==
==Enzyme Mechanism==
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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-postition oxygen of the steroid, thereby facilitating the abstraction of the &beta;-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>
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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 &beta;-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.
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.
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===Low Barrier Hydrogen Bond===
===Low Barrier Hydrogen Bond===
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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> 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" />
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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" />
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====LBHB Controversy====
+
===LBHB Controversy and Catalytic Efficiency from Preorganized State===
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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_2_1qjg/1'>Model 2</scene>.<ref name="Kraut2010" />,<ref name="Sigala2009">PMID:19469568 </ref> Additionally, he suggests that hydrogen bonding is important within the active site of KSI albeit from an entropic rather than energetic perspective.<ref name="Kraut2006">PMID:16602823 </ref> 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 within the enzyme active site.<ref name="Kraut2006" />
+
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 from an entropic rather than energetic perspective.<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 within the enzyme active site.<ref name="Kraut2006" />
-
==Implications of Hydrophobic Active Site==
+
===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. To prevent divergence of Asp<sup>38</sup> from this ''normal'' pKa during initial substrate binding, it is hydrogen bonded to two or three water molecules which are excluded from the active site later in the enzymatic reaction. Maintenance of this ''normal'' pKa is important because protonation of Asp<sup>38</sup> would no longer permit it to carry out hydrogen abstraction.<ref name="Pollack" />
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. To prevent divergence of Asp<sup>38</sup> from this ''normal'' pKa during initial substrate binding, it is hydrogen bonded to two or three water molecules which are excluded from the active site later in the enzymatic reaction. Maintenance of this ''normal'' pKa is important because protonation of Asp<sup>38</sup> would no longer permit it to carry out hydrogen abstraction.<ref name="Pollack" />

Revision as of 03:03, 3 May 2010

Contents

Ketosteroid Isomerase

Introduction

Template:STRUCTURE 1isk (KSI, EC#5.3.3.1) is an enzyme that catalyzes the isomerization of 3-oxo-Δ5 ketosteroids to their hormonally active Δ4-conjugated isomers, as illustrated below.[1], [2]

This reaction is essential in the biosynthesis of steroids in mammals where KSI is a membrane-bound complex.[3] In bacteria, however, KSI exists as a soluble protein is involves in catabolism of steroids.[3] It was first isolated in and has been extensively studied in Commamonas tetosteroni (TI), a bacteria that is capable of growth with testosterone as its sole carbon source.[4] Structural and kinetic studies of this and its homolog from Pseudomonas putida with which it shares 34% sequence and near identical structural homology.[1],[3] It is one of the most efficient known enzymes with an essentially diffusion limited rate of catalysis.[2],[5] It is capable of increasing the catalytic rate by eleven orders of magnitude.[6] Its high catalytic efficiency and unique active site geometry have made it fertile ground for examining the validity of the low barrier hydrogen bond hypothesis[7] and electrostatic preorganization.[8].

Structure

Ketosteroid isomerase exits as a 28 kDa homodimeric protein, in which the two dimers related to each other via hydrophobic and electrostatic interactions.[5] Each monomer consists of a curved and three . These secondary structures define a conical closed barrel geometry, with one open and one closed end, and create a deep pocket in which the active site resides.[3],[9] This unique geometry is shared by several other proteins (scytalone dehydratase, nuclear transport factor 2, and naphthalene 1,2-dioxygenase), however, these molecules do not share functional or sequence homology. It is speculated that this unique protein structure may enable better binding of hydrophobic substrates such as steroids.[3]

Although the of KSI is notably , 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.[1],[10] Additionally, the active site contains an aspartate residue at position 38 that is participates in the catalytic activity of KSI.[1]

Enzyme Mechanism

A monomer of ketosteroid isomerase is shown in complex with the transition state analog equinelin (PBD Structure ID 1QJG).

Drag the structure with the mouse to rotate

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.[1] 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.[1],[3] Structural and kinetic studies suggest that Asp38 (numbering is that of the TI varient of KSI) serves as a general base in this reaction as shown below and abstracts the %beta;-proton from C4 with the syn orbitals of its carboxylate group.[5] Note the formation of the unstable enolate intermediate.

Tyr14 and Asp99 are believed to participate in hydrogen bonding to the O-3 carbonyl of the substrate steroid and stabilize reaction intermediates. Tyr14 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 , Tyr14 and Asp99 are both bound to the 3-position oxygen, whereas, in , they form a hydrogen bonding network. These are as a single monomer in complex with the intermediate analog equilenin.[11]

Model 1 has become the generally accepted scheme through both crystallographic and mutanagenic/kinetic approaches. Mutation of Asp99 to alanine and Tyr14 to phenylalanine resulted in deleterious effects on kinetic parameters, which were additive in nature suggesting that Tyr14 and Asp99 participate equally in hydrogen binding with the oxygen atom. Additionally, the crystal structure of TI analog from Pseudomonas putida supports this conclusion given the .[1] The general mechanism in the active site of KSI is outlined below.

Low Barrier Hydrogen Bond

Ketosteroid isomerase has become a test enzyme in the debate over the existence of low barrier hydrogen bonds (LBHBs) in accordance with the hydrogen bonding discussed above. 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 Tyr14 and the O-3 atom of the analogs and suggestive of the formation of a bridging hydrogen with short bonds.[3],[12],[7] Additionally, NMR fractionation studies with deuterium substitution are strongly suggestive of the high strength of this bond as deuterium is retained preferentially in this position.[12] The energy of this bond along with that of the normal hydrogen bond from Asp99 most likely contribute to the energy needed to support proton abstraction from the C-4 position.[3]

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[13], a viewpoint that is shared among a subset of enzymologists.[14] Hershlag and coworkers are proponents of the hydrogen bonding mechanism described in ;[14],[15] however, he suggests that hydrogen bonding is important within the active site of KSI albeit from an entropic rather than energetic perspective.[8] 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 within the enzyme active site.[8]

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 acetate anion is used to mimic the functionality of Asp38 and has a similar pKaof 4.6. To prevent divergence of Asp38 from this normal pKa during initial substrate binding, it is hydrogen bonded to two or three water molecules which are excluded from the active site later in the enzymatic reaction. Maintenance of this normal pKa is important because protonation of Asp38 would no longer permit it to carry out hydrogen abstraction.[1]

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 mandelate racemace, triosephosphate isomerase, citrate synthase, and 4-oxalocrotonate tautomerase.[1]

Structural Homologs

As noted above, scytalone dehydratase, nuclear transport factor 2 (NTF2), and naphthalene 1,2-dioxygenase share similar structural motifs to ketosteroid isomerase, which facilitate binding of hydrophobic substrates.[3],[6] 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.[6] In a recent paper, Cherney et al[16]. 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.[16]

Available Structures

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Pollack RM. Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase. Bioorg Chem. 2004 Oct;32(5):341-53. PMID:15381400 doi:10.1016/j.bioorg.2004.06.005
  2. ↑ 2.0 2.1 TALALAY P, WANG VS. Enzymic isomerization of delta5-3-ketosteroids. Biochim Biophys Acta. 1955 Oct;18(2):300-1. PMID:13276386
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 Ha NC, Choi G, Choi KY, Oh BH. Structure and enzymology of Delta5-3-ketosteroid isomerase. Curr Opin Struct Biol. 2001 Dec;11(6):674-8. PMID:11751047
  4. ↑ Ha NC, Choi G, Choi KY, Oh BH. Structure and enzymology of Delta5-3-ketosteroid isomerase. Curr Opin Struct Biol. 2001 Dec;11(6):674-8. PMID:11751047
  5. ↑ 5.0 5.1 5.2 Wu ZR, Ebrahimian S, Zawrotny ME, Thornburg LD, Perez-Alvarado GC, Brothers P, Pollack RM, Summers MF. Solution structure of 3-oxo-delta5-steroid isomerase. Science. 1997 Apr 18;276(5311):415-8. PMID:9103200
  6. ↑ 6.0 6.1 6.2 Murzin AG. How far divergent evolution goes in proteins. Curr Opin Struct Biol. 1998 Jun;8(3):380-7. PMID:9666335
  7. ↑ 7.0 7.1 Cleland WW, Frey PA, Gerlt JA. The low barrier hydrogen bond in enzymatic catalysis. J Biol Chem. 1998 Oct 2;273(40):25529-32. PMID:9748211
  8. ↑ 8.0 8.1 8.2 Kraut DA, Sigala PA, Pybus B, Liu CW, Ringe D, Petsko GA, Herschlag D. Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole. PLoS Biol. 2006 Apr;4(4):e99. Epub 2006 Mar 28. PMID:16602823 doi:10.1371/journal.pbio.0040099
  9. ↑ Cho HS, Choi G, Choi KY, Oh BH. Crystal structure and enzyme mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni. Biochemistry. 1998 Jun 9;37(23):8325-30. PMID:9622484 doi:10.1021/bi9801614
  10. ↑ Sigala PA, Kraut DA, Caaveiro JM, Pybus B, Ruben EA, Ringe D, Petsko GA, Herschlag D. Testing geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole. J Am Chem Soc. 2008 Oct 15;130(41):13696-708. Epub 2008 Sep 23. PMID:18808119 doi:10.1021/ja803928m
  11. ↑ Cho HS, Ha NC, Choi G, Kim HJ, Lee D, Oh KS, Kim KS, Lee W, Choi KY, Oh BH. Crystal structure of delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization. J Biol Chem. 1999 Nov 12;274(46):32863-8. PMID:10551849
  12. ↑ 12.0 12.1 Zhao Q, Abeygunawardana C, Talalay P, Mildvan AS. NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8220-4. PMID:8710850
  13. ↑ Zhao Q, Abeygunawardana C, Talalay P, Mildvan AS. NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8220-4. PMID:8710850
  14. ↑ 14.0 14.1 Kraut DA, Sigala PA, Fenn TD, Herschlag D. Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):1960-5. Epub 2010 Jan 11. PMID:20080683
  15. ↑ Sigala PA, Caaveiro JM, Ringe D, Petsko GA, Herschlag D. Hydrogen bond coupling in the ketosteroid isomerase active site. Biochemistry. 2009 Jul 28;48(29):6932-9. PMID:19469568 doi:10.1021/bi900713j
  16. ↑ 16.0 16.1 Cherney MM, Garen CR, James MN. Crystal structure of Mycobacterium tuberculosis Rv0760c at 1.50 A resolution, a structural homolog of Delta(5)-3-ketosteroid isomerase. Biochim Biophys Acta. 2008 Nov;1784(11):1625-32. Epub 2008 Jun 6. PMID:18589008 doi:10.1016/j.bbapap.2008.05.012

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