2WUD

Crystal structure of S114A mutant of HsaD from Mycobacterium tuberculosis


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.239 
  • R-Value Work: 0.192 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Characterization of a carbon-carbon hydrolase from Mycobacterium tuberculosis involved in cholesterol metabolism.

Lack, N.A.Yam, K.C.Lowe, E.D.Horsman, G.P.Owen, R.L.Sim, E.Eltis, L.D.

(2010) J Biol Chem 285: 434-443

  • DOI: https://doi.org/10.1074/jbc.M109.058081
  • Primary Citation of Related Structures:  
    2WUD, 2WUE, 2WUF, 2WUG

  • PubMed Abstract: 

    In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4-33 times higher specificity for DSHA (k(cat)/K(m) = 3.3 +/- 0.3 x 10(4) m(-1) s(-1)) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K(d) of 51 +/- 2 mum. The S114A.DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8-1.9 Aindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the alpha-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 A, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.


  • Organizational Affiliation

    Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
2-HYDROXY-6-OXO-6-PHENYLHEXA-2,4-DIENOATE HYDROLASE BPHD
A, B
291Mycobacterium tuberculosis H37RvMutation(s): 1 
EC: 3.7.1.8
UniProt
Find proteins for P9WNH5 (Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv))
Explore P9WNH5 
Go to UniProtKB:  P9WNH5
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP9WNH5
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.239 
  • R-Value Work: 0.192 
  • Space Group: F 2 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 112.254α = 90
b = 118.765β = 90
c = 182.592γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
SAINTdata reduction
SADABSdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2009-10-20
    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: 2017-07-05
    Changes: Data collection
  • Version 1.4: 2018-02-28
    Changes: Database references, Source and taxonomy
  • Version 1.5: 2023-12-20
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description