3AFE

Crystal structure of the HsaA monooxygenase from M.tuberculosis


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.191 
  • R-Value Observed: 0.194 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism

Dresen, C.Lin, L.Y.D'Angelo, I.Tocheva, E.I.Strynadka, N.Eltis, L.D.

(2010) J Biol Chem 285: 22264-22275

  • DOI: https://doi.org/10.1074/jbc.M109.099028
  • Primary Citation of Related Structures:  
    3AFE, 3AFF

  • PubMed Abstract: 

    Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.


  • Organizational Affiliation

    Department of Microbiology and Immunology, University of British Columbia, Vancouver V6T 1Z3, Canada.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Hydroxylase, putative
A, B, C, D
394Mycobacterium tuberculosisMutation(s): 0 
UniProt
Find proteins for P9WJA1 (Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv))
Explore P9WJA1 
Go to UniProtKB:  P9WJA1
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP9WJA1
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.191 
  • R-Value Observed: 0.194 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 90.968α = 90
b = 94.009β = 93.94
c = 98.094γ = 90
Software Package:
Software NamePurpose
MAR345dtbdata collection
MOLREPphasing
PHENIXrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-05-26
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2023-11-01
    Changes: Data collection, Database references, Refinement description