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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
Author(s) -
Jensen Chantel N.,
Mielke Tamara,
Farrugia Joseph E.,
Frank Annika,
Man Henry,
Hart Sam,
Turkenburg Johan P.,
Grogan Gideon
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402701
Subject(s) - monooxygenase , hydroxylation , stereochemistry , biochemistry , chemistry , mutant , active site , residue (chemistry) , enzyme , directed evolution , enzyme kinetics , oxygenase , biology , gene , cytochrome p450
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.