1OGZ

Crystal Structure Of 5-3-Ketosteroid Isomerase Mutants P39A Complexed With Equilenin From Pseudomonas Testosteroni


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.30 Å
  • R-Value Free: 0.309 
  • R-Value Work: 0.235 
  • R-Value Observed: 0.235 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

The Conserved Cis-Pro39 Residue Plays a Crucial Role in the Proper Positioning of the Catalytic Base Asp38 in Ketosteroid Isomerase from Comamonas Testosteroni.

Nam, G.H.Cha, S.Yun, Y.S.Oh, Y.H.Hong, B.H.Lee, H.Choi, K.Y.

(2003) Biochem J 375: 297

  • DOI: https://doi.org/10.1042/BJ20030263
  • Primary Citation of Related Structures:  
    1OGZ

  • PubMed Abstract: 

    KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers at a reaction rate equivalent to the diffusion-controlled limit. Based on the structural analysis of KSI at a high resolution, the conserved cis-Pro39 residue was proposed to be involved in the proper positioning of Asp38, a critical catalytic residue, since the residue was found not only to be structurally associated with Asp38, but also to confer a structural rigidity on the local active-site geometry consisting of Asp38, Pro39, Val40, Gly41 and Ser42 at the flexible loop between b-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro39 residue near the active site of KSI, Pro39 was replaced with alanine or glycine. The free energy of activation for the P39A and P39G mutants increased by 10.5 and 16.7 kJ/mol (2.5 and 4.0 kcal/mol) respectively, while DG(U)H2O (the free-energy change for unfolding in the absence of urea at 25.00+/-0.02 degrees C) decreased by 31.0 and 35.6 kJ/mol (7.4 and 8.5 kcal/mol) respectively, compared with the wild-type enzyme. The crystal structure of the P39A mutant in complex with d-equilenin [d-1,3,5(10),6,8-estrapentaen-3-ol-17-one], a reaction intermediate analogue, determined at 2.3 A (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp38, as well as the local active-site geometry near Asp38, which resulted in substantial decreases in the activity and stability of KSI. Upon binding 1-anilinonaphthalene-8-sulphonic acid, the fluorescence intensities of the P39A and P39G mutants were increased drastically, with maximum wavelengths blue-shifted upon binding, indicating that the mutations might alter the hydrophobic active site of KSI. Taken together, our results demonstrate that the conserved cis-Pro39 residue plays a crucial role in the proper positioning of the critical catalytic base Asp38 and in the structural integrity of the active site in KSI.


  • Organizational Affiliation

    National Research Laboratory of Protein Folding and Engineering, Pohang 790-784, Republic of Korea.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
STEROID DELTA-ISOMERASE125Comamonas 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
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
EQU
Query on EQU

Download Ideal Coordinates CCD File 
B [auth A]EQUILENIN
C18 H18 O2
PDRGHUMCVRDZLQ-WMZOPIPTSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
EQU PDBBind:  1OGZ Kd: 1400 (nM) from 1 assay(s)
Binding MOAD:  1OGZ Kd: 1400 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.30 Å
  • R-Value Free: 0.309 
  • R-Value Work: 0.235 
  • R-Value Observed: 0.235 
  • Space Group: P 65 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 60.733α = 90
b = 60.733β = 90
c = 142.803γ = 120
Software Package:
Software NamePurpose
CNSrefinement

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-09-04
    Type: Initial release
  • Version 1.1: 2011-05-08
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance