Structures of the Apo and FAD-bound forms of 2-hydroxybiphenyl 3-monooxygenase (HbpA) locate activity hotspots identified by using directed evolution.
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.
York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, YO10 5DD (UK).
Chantel N
CN
Jensen
Tamara
T
Mielke
Joseph E
JE
Farrugia
Annika
A
Frank
Henry
H
Man
Sam
S
Hart
Johan P
JP
Turkenburg
Gideon
G
Grogan
D001051:Apoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001713:Biphenyl Compounds / Q000378:metabolism; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011549:Pseudomonas / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D001051:Apoenzymes; D001713:Biphenyl Compounds; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C004369:2-phenylphenol; D006899:Mixed Function Oxygenases; C058145:2-hydroxybiphenyl 3-monooxygenase
FAD; biotransformations; flavoprotein; hydroxylation; monooxygenases