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PDBsum entry 1opy
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Isomerase PDB id
1opy

 

 

 

 

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Contents
Protein chain
123 a.a. *
Waters ×48
* Residue conservation analysis
PDB id:
1opy
Name: Isomerase
Title: Ksi
Structure: Delta5-3-ketosteroid iosmerase. Chain: a. Synonym: ksi, 3-ketosteroid isomerase. Engineered: yes
Source: Pseudomonas putida. Organism_taxid: 303. Strain: biotype b. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
Resolution:
1.90Å     R-factor:   0.199     R-free:   0.273
Authors: S.-W.Kim,S.-S.Cha,H.-S.Cho,J.-S.Kim,N.-C.Ha,M.-J.Cho,K.-Y.Choi,B.- H.Oh
Key ref:
S.W.Kim et al. (1997). High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue. Biochemistry, 36, 14030-14036. PubMed id: 9369474 DOI: 10.1021/bi971546+
Date:
23-May-97     Release date:   08-Apr-98    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P07445  (SDIS_PSEPU) -  Steroid Delta-isomerase from Pseudomonas putida
Seq:
Struc: 		131 a.a.
123 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.5.3.3.1  - steroid Delta-isomerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: a 3-oxo-Delta5-steroid = a 3-oxo-Delta4-steroid
3-oxo-Delta(5)-steroid
 = 3-oxo-Delta(4)-steroid
Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 

 
    Added reference    
 
 
DOI no: 10.1021/bi971546+ Biochemistry 36:14030-14036 (1997)
PubMed id: 9369474  
 
 
High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue.
S.W.Kim, S.S.Cha, H.S.Cho, J.S.Kim, N.C.Ha, M.J.Cho, S.Joo, K.K.Kim, K.Y.Choi, B.H.Oh.
 
  ABSTRACT  
 
Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
20080683 D.A.Kraut, P.A.Sigala, T.D.Fenn, and D.Herschlag (2010).
Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole.
  Proc Natl Acad Sci U S A, 107, 1960-1965.
PDB code: 3ipt
20143849 W.Childs, and S.G.Boxer (2010).
Proton affinity of the oxyanion hole in the active site of ketosteroid isomerase.
  Biochemistry, 49, 2725-2731.  
20397697 W.Childs, and S.G.Boxer (2010).
Solvation response along the reaction coordinate in the active site of ketosteroid isomerase.
  J Am Chem Soc, 132, 6474-6480.  
19301315 C.Li, K.E.Roege, and W.L.Kelly (2009).
Analysis of the indanomycin biosynthetic gene cluster from Streptomyces antibioticus NRRL 8167.
  Chembiochem, 10, 1064-1072.  
  19260691 D.A.Kraut, M.J.Churchill, P.E.Dawson, and D.Herschlag (2009).
Evaluating the potential for halogen bonding in the oxyanion hole of ketosteroid isomerase using unnatural amino acid mutagenesis.
  ACS Chem Biol, 4, 269-273.  
19799395 D.K.Chakravorty, A.V.Soudackov, and S.Hammes-Schiffer (2009).
Hybrid quantum/classical molecular dynamics simulations of the proton transfer reactions catalyzed by ketosteroid isomerase: analysis of hydrogen bonding, conformational motions, and electrostatics.
  Biochemistry, 48, 10608-10619.  
19846764 E.W.Debler, R.Müller, D.Hilvert, and I.A.Wilson (2009).
An aspartate and a water molecule mediate efficient acid-base catalysis in a tailored antibody pocket.
  Proc Natl Acad Sci U S A, 106, 18539-18544.
PDB codes: 3fo0 3fo1 3fo2
19706511 J.P.Schwans, D.A.Kraut, and D.Herschlag (2009).
Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase.
  Proc Natl Acad Sci U S A, 106, 14271-14275.  
18004666 H.J.Lee, H.S.Moon, d.o. .S.Jang, H.J.Cha, B.H.Hong, K.Y.Choi, and H.C.Lee (2008).
Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H-15N multidimensional NMR spectroscopy.
  J Biomol NMR, 40, 65-70.  
18442985 H.J.Lee, d.o. .S.Jang, H.J.Cha, H.S.Moon, B.H.Hong, K.Y.Choi, and H.C.Lee (2008).
NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea.
  J Biochem, 144, 215-221.  
16632258 A.R.Gallimore, C.B.Stark, A.Bhatt, B.M.Harvey, Y.Demydchuk, V.Bolanos-Garcia, D.J.Fowler, J.Staunton, P.F.Leadlay, and J.B.Spencer (2006).
Evidence for the role of the monB genes in polyether ring formation during monensin biosynthesis.
  Chem Biol, 13, 453-460.  
16602823 D.A.Kraut, P.A.Sigala, B.Pybus, C.W.Liu, D.Ringe, G.A.Petsko, and D.Herschlag (2006).
Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole.
  PLoS Biol, 4, e99.
PDB codes: 2b32 2pzv
16889958 V.Z.Pletnev, J.L.Thomas, F.L.Rhaney, L.S.Holt, L.A.Scaccia, T.C.Umland, and W.L.Duax (2006).
Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1.
  J Steroid Biochem Mol Biol, 101, 50-60.  
16040747 M.J.Yonkunas, Y.Xu, and P.Tang (2005).
Anesthetic interaction with ketosteroid isomerase: insights from molecular dynamics simulations.
  Biophys J, 89, 2350-2356.  
15819891 Y.S.Yun, G.H.Nam, Y.G.Kim, B.H.Oh, and K.Y.Choi (2005).
Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B.
  FEBS J, 272, 1999-2011.
PDB code: 1w6y
15071504 A.Sultana, P.Kallio, A.Jansson, J.S.Wang, J.Niemi, P.Mäntsälä, and G.Schneider (2004).
Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation.
  EMBO J, 23, 1911-1921.
PDB code: 1sjw
15159577 E.G.Ahuja, D.V.Mavrodi, L.S.Thomashow, and W.Blankenfeldt (2004).
Overexpression, purification and crystallization of PhzA, the first enzyme of the phenazine biosynthesis pathway of Pseudomonas fluorescens 2-79.
  Acta Crystallogr D Biol Crystallogr, 60, 1129-1131.  
12773375 M.Arand, B.M.Hallberg, J.Zou, T.Bergfors, F.Oesch, M.J.van der Werf, J.A.de Bont, T.A.Jones, and S.L.Mowbray (2003).
Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site.
  EMBO J, 22, 2583-2592.
PDB codes: 1nu3 1nww
12734184 Y.S.Yun, T.H.Lee, G.H.Nam, D.S.Jang, S.Shin, B.H.Oh, and K.Y.Choi (2003).
Origin of the different pH activity profile in two homologous ketosteroid isomerases.
  J Biol Chem, 278, 28229-28236.
PDB code: 1ocv
11872752 A.S.Johansson, and B.Mannervik (2002).
Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3.
  J Biol Chem, 277, 16648-16654.  
11274465 D.H.Kim, G.H.Nam, D.S.Jang, S.Yun, G.Choi, H.C.Lee, and K.Y.Choi (2001).
Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B.
  Protein Sci, 10, 741-752.  
11389596 G.Choi, N.C.Ha, M.S.Kim, B.H.Hong, B.H.Oh, and K.Y.Choi (2001).
Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.
  Biochemistry, 40, 6828-6835.
PDB codes: 1e97 1ea2
10963661 B.Golinelli-Pimpaneau, O.Goncalves, T.Dintinger, D.Blanchard, M.Knossow, and C.Tellier (2000).
Structural evidence for a programmed general base in the active site of a catalytic antibody.
  Proc Natl Acad Sci U S A, 97, 9892-9895.
PDB code: 1f3d
10769113 D.H.Kim, D.S.Jang, G.H.Nam, G.Choi, J.S.Kim, N.C.Ha, M.S.Kim, B.H.Oh, and K.Y.Choi (2000).
Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B.
  Biochemistry, 39, 4581-4589.
PDB codes: 1dmm 1dmn 1dmq
11041875 D.H.Kim, D.S.Jang, G.H.Nam, S.Yun, J.H.Cho, G.Choi, H.C.Lee, and K.Y.Choi (2000).
Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni.
  Biochemistry, 39, 13084-13092.  
10653633 G.Choi, N.C.Ha, S.W.Kim, D.H.Kim, S.Park, B.H.Oh, and K.Y.Choi (2000).
Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.
  Biochemistry, 39, 903-909.
PDB code: 1cqs
10675319 I.Grishkovskaya, G.V.Avvakumov, G.Sklenar, D.Dales, G.L.Hammond, and Y.A.Muller (2000).
Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain.
  EMBO J, 19, 504-512.
PDB code: 1d2s
10841545 K.S.Kim, K.S.Oh, and J.Y.Lee (2000).
Catalytic role of enzymes: short strong H-bond-induced partial proton shuttles and charge redistributions.
  Proc Natl Acad Sci U S A, 97, 6373-6378.  
11076530 K.S.Oh, S.S.Cha, D.H.Kim, H.S.Cho, N.C.Ha, G.Choi, J.Y.Lee, P.Tarakeshwar, H.S.Son, K.Y.Choi, B.H.Oh, and K.S.Kim (2000).
Role of catalytic residues in enzymatic mechanisms of homologous ketosteroid isomerases.
  Biochemistry, 39, 13891-13896.  
10551849 H.S.Cho, N.C.Ha, G.Choi, H.J.Kim, D.Lee, K.S.Oh, K.S.Kim, W.Lee, K.Y.Choi, and B.H.Oh (1999).
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, 274, 32863-32868.
PDB code: 1qjg
10328262 T.K.Harris, and A.S.Mildvan (1999).
High-precision measurement of hydrogen bond lengths in proteins by nuclear magnetic resonance methods.
  Proteins, 35, 275-282.  
10382670 Z.Wawrzak, T.Sandalova, J.J.Steffens, G.S.Basarab, T.Lundqvist, Y.Lindqvist, and D.B.Jordan (1999).
High-resolution structures of scytalone dehydratase-inhibitor complexes crystallized at physiological pH.
  Proteins, 35, 425-439.
PDB codes: 4std 5std 6std 7std
9778344 A.B.Taylor, R.M.Czerwinski, W.H.Johnson, C.P.Whitman, and M.L.Hackert (1998).
Crystal structure of 4-oxalocrotonate tautomerase inactivated by 2-oxo-3-pentynoate at 2.4 A resolution: analysis and implications for the mechanism of inactivation and catalysis.
  Biochemistry, 37, 14692-14700.
PDB code: 1bjp
9666335 A.G.Murzin (1998).
How far divergent evolution goes in proteins.
  Curr Opin Struct Biol, 8, 380-387.  
9657686 B.Brooks, R.S.Phillips, and W.F.Benisek (1998).
High-efficiency incorporation in vivo of tyrosine analogues with altered hydroxyl acidity in place of the catalytic tyrosine-14 of Delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: effects of the modifications on isomerase kinetics.
  Biochemistry, 37, 9738-9742.  
9622484 H.S.Cho, G.Choi, K.Y.Choi, and B.H.Oh (1998).
Crystal structure and enzyme mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni.
  Biochemistry, 37, 8325-8330.
PDB code: 8cho
9718318 J.Lin, C.S.Cassidy, and P.A.Frey (1998).
Correlations of the basicity of His 57 with transition state analogue binding, substrate reactivity, and the strength of the low-barrier hydrogen bond in chymotrypsin.
  Biochemistry, 37, 11940-11948.  
9657682 J.M.Jez, and T.M.Penning (1998).
Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase.
  Biochemistry, 37, 9695-9703.  
9671521 L.D.Thornburg, F.Hénot, D.P.Bash, D.C.Hawkinson, S.D.Bartel, and R.M.Pollack (1998).
Electrophilic assistance by Asp-99 of 3-oxo-Delta 5-steroid isomerase.
  Biochemistry, 37, 10499-10506.  
9578560 L.Qi, and R.M.Pollack (1998).
Catalytic contribution of phenylalanine-101 of 3-oxo-Delta 5-steroid isomerase.
  Biochemistry, 37, 6760-6766.  
9778345 M.A.Massiah, C.Abeygunawardana, A.G.Gittis, and A.S.Mildvan (1998).
Solution structure of Delta 5-3-ketosteroid isomerase complexed with the steroid 19-nortestosterone hemisuccinate.
  Biochemistry, 37, 14701-14712.
PDB code: 1buq
9739087 Y.Modis, S.A.Filppula, D.K.Novikov, B.Norledge, J.K.Hiltunen, and R.K.Wierenga (1998).
The crystal structure of dienoyl-CoA isomerase at 1.5 A resolution reveals the importance of aspartate and glutamate sidechains for catalysis.
  Structure, 6, 957-970.
PDB code: 1dci
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.

 

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