2OG1

Crystal Structure of BphD, a C-C hydrolase from Burkholderia xenovorans LB400


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Free: 0.224 
  • R-Value Work: 0.208 

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This is version 1.4 of the entry. See complete history


Literature

Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway

Horsman, G.P.Ke, J.Dai, S.Seah, S.Y.Bolin, J.T.Eltis, L.D.

(2006) Biochemistry 45: 11071-11086

  • DOI: https://doi.org/10.1021/bi0611098
  • Primary Citation of Related Structures:  
    2OG1

  • PubMed Abstract: 

    Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.


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
A, B
286Paraburkholderia xenovorans LB400Mutation(s): 0 
Gene Names: bphD
EC: 3.7.1.8
UniProt
Find proteins for P47229 (Paraburkholderia xenovorans (strain LB400))
Explore P47229 
Go to UniProtKB:  P47229
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP47229
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.60 Å
  • R-Value Free: 0.224 
  • R-Value Work: 0.208 
  • Space Group: P 64
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 135α = 90
b = 135β = 90
c = 66.725γ = 120
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
SOLVEphasing
CNSrefinement
PDB_EXTRACTdata extraction
ADSCdata collection

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2007-01-16
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Source and taxonomy, Version format compliance
  • Version 1.3: 2017-10-18
    Changes: Refinement description
  • Version 1.4: 2023-12-27
    Changes: Data collection, Database references, Derived calculations