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PDBsum entry 1ocv

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protein Protein-protein interface(s) links
Ketosteroid isomerase PDB id
1ocv
Jmol
Contents
Protein chains
125 a.a. *
Waters ×357
* Residue conservation analysis
PDB id:
1ocv
Name: Ketosteroid isomerase
Title: The f116w mutant structure of ketosteroid isomerase from comamonas testosteroni
Structure: Steroid delta-isomerase. Chain: a, b, c, d. Synonym: ketosteroid isomerase. Engineered: yes. Mutation: yes
Source: Comamonas testosteroni. Organism_taxid: 285. Expressed in: escherichia coli. Expression_system_taxid: 469008.
Biol. unit: Dimer (from PDB file)
Resolution:
2.0Å     R-factor:   0.228     R-free:   0.282
Authors: Y.S.Yun,T.-H.Lee,S.Shin
Key ref:
Y.S.Yun et al. (2003). Origin of the different pH activity profile in two homologous ketosteroid isomerases. J Biol Chem, 278, 28229-28236. PubMed id: 12734184 DOI: 10.1074/jbc.M302166200
Date:
11-Feb-03     Release date:   24-Jul-03    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P00947  (SDIS_COMTE) -  Steroid Delta-isomerase
Seq:
Struc:
125 a.a.
125 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 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
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     intracellular   1 term 
  Biological process     transport   3 terms 
  Biochemical function     isomerase activity     2 terms  

 

 
    Added reference    
 
 
DOI no: 10.1074/jbc.M302166200 J Biol Chem 278:28229-28236 (2003)
PubMed id: 12734184  
 
 
Origin of the different pH activity profile in two homologous ketosteroid isomerases.
Y.S.Yun, T.H.Lee, G.H.Nam, d.o. .S.Jang, S.Shin, B.H.Oh, K.Y.Choi.
 
  ABSTRACT  
 
Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.
 
  Selected figure(s)  
 
Figure 2.
FIG. 2. pH dependence of kinetic parameters of WT and mutant KSIs. The lines represent nonlinear least-squares fits of the data to Equations 1 and 2 to obtain the pK[E] and pK[ES] values, respectively, as listed in Table I. 5,10-EST was used as a substrate for graphs C and D, and for PI-WT in graphs A and B. 5-AND was used as a substrate for graphs E and F, and for PI-W116F in graphs A and B.
Figure 4.
FIG. 4. Stereoview of the catalytic base of TI-F116W with 2F[o]-F[c]-simulated annealing omit electron density map contoured at 1.0 . Residues Trp-116 and Asp-38, which were omitted from the model, display clear electron density. A hydrogen bond between Asp-38 and Trp-116 is represented by a dashed line. The figure was drawn by using the program BobScript and rendered using Raster3D.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 28229-28236) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
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
14566064 S.E.Iismaa, S.Holman, M.A.Wouters, L.Lorand, R.M.Graham, and A.Husain (2003).
Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases.
  Proc Natl Acad Sci U S A, 100, 12636-12641.  
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