1OCV

the F116W mutant structure of ketosteroid isomerase from Comamonas testosteroni


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.282 
  • R-Value Work: 0.229 
  • R-Value Observed: 0.229 

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This is version 1.3 of the entry. See complete history


Literature

Origin of the Different Ph Activity Profile in Two Homologous Ketosteroid Isomerases

Yun, Y.S.Lee, T.-H.Nam, G.H.Jang, D.S.Shin, S.Oh, B.-H.Choi, K.Y.

(2003) J Biol Chem 278: 28229

  • DOI: https://doi.org/10.1074/jbc.M302166200
  • Primary Citation of Related Structures:  
    1OCV

  • PubMed 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.


  • Organizational Affiliation

    Division of Molecular and Life Sciences, the National Research Laboratory of Protein Folding and Engineering, Pohang University of Science and Technology, Pohang 790-784, South Korea.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
STEROID DELTA-ISOMERASE
A, B, C, D
125Comamonas testosteroniMutation(s): 1 
EC: 5.3.3.1
UniProt
Find proteins for P00947 (Comamonas testosteroni)
Explore P00947 
Go to UniProtKB:  P00947
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00947
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.282 
  • R-Value Work: 0.229 
  • R-Value Observed: 0.229 
  • Space Group: P 31
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 71.525α = 90
b = 71.525β = 90
c = 103.34γ = 120
Software Package:
Software NamePurpose
CNSrefinement
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-07-24
    Type: Initial release
  • Version 1.1: 2011-05-08
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2023-12-13
    Changes: Data collection, Database references, Other, Refinement description