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Structures of the Apo and FAD-Bound Forms of 2-Hydroxybiphenyl 3-monooxygenase (HbpA) Locate Activity Hotspots Identified by Using Directed Evolution

DOI: 10.1002/cbic.201402701 DOI Help
PMID: 25737306 PMID Help

Authors: Chantel N. Jensen (University of York) , Tamara Mielke (University of York) , Joseph E. Farrugia (University of York) , Annika Frank (University of York) , Henry Man (University of York) , Sam Hart (University of York) , Johan Turkenburg (University of York) , Gideon Grogan (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chembiochem

State: Published (Approved)
Published: March 2015
Diamond Proposal Number(s): 7864

Open Access Open Access

Abstract: The FAD-dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2-hydroxybiphenyl (2HBP) to 2,3-dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed-evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD-complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate-binding site.

Journal Keywords: Biotransformations; Fad; Flavoprotein; Hydroxylation; Monooxygenases

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography

Added On: 27/03/2015 13:22

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