4QCK

Crystal structure of 3-ketosteroid-9-alpha-hydroxylase (KshA) from M. tuberculosis in complex with 4-androstene-3,17-dione


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.46 Å
  • R-Value Free: 0.235 
  • R-Value Work: 0.177 
  • R-Value Observed: 0.180 

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This is version 1.2 of the entry. See complete history


Literature

Substrate specificities and conformational flexibility of 3-ketosteroid 9 alpha-hydroxylases.

Penfield, J.S.Worrall, L.J.Strynadka, N.C.Eltis, L.D.

(2014) J Biol Chem 289: 25523-25536

  • DOI: https://doi.org/10.1074/jbc.M114.575886
  • Primary Citation of Related Structures:  
    4QCK, 4QDC, 4QDD, 4QDF

  • PubMed Abstract: 

    KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >10(5) s(-1) M(-1) for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (Ki S ∼100 μM). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue "mouth loop," which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.


  • Organizational Affiliation

    From the Departments of Biochemistry and Molecular Biology and.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
3-ketosteroid-9-alpha-monooxygenase oxygenase subunit386Mycobacterium tuberculosis H37RvMutation(s): 0 
Gene Names: kshARv3526
EC: 1.14.13.142 (PDB Primary Data), 1.14.15.30 (UniProt)
UniProt
Find proteins for P71875 (Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv))
Explore P71875 
Go to UniProtKB:  P71875
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP71875
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.46 Å
  • R-Value Free: 0.235 
  • R-Value Work: 0.177 
  • R-Value Observed: 0.180 
  • Space Group: P 3 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 115.8α = 90
b = 115.8β = 90
c = 80.81γ = 120
Software Package:
Software NamePurpose
XSCALEdata scaling
PHASERphasing
REFMACrefinement
PDB_EXTRACTdata extraction

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2014-07-30
    Type: Initial release
  • Version 1.1: 2014-10-01
    Changes: Database references
  • Version 1.2: 2024-02-28
    Changes: Data collection, Database references, Derived calculations