title: Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias.
abstract: Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such "gatekeeper" mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme.
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom and.
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Butler
authors.affiliation: Cypex Ltd., 6 Tom McDonald Avenue, Dundee DD2 1NH, Scotland, United Kingdom.
authors.forename: Caroline
authors.initials: C
authors.lastname: Peet
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom and.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Mason
authors.affiliation: Cypex Ltd., 6 Tom McDonald Avenue, Dundee DD2 1NH, Scotland, United Kingdom.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Voice
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom and.
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.affiliation: From the Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom and. Electronic address: Andrew.Munro@Manchester.ac.uk.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation / Q000235:genetics; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009853:Omeprazole / Q000737:chemistry* / Q000493:pharmacokinetics; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D054328:Proton Pump Inhibitors / Q000737:chemistry* / Q000493:pharmacokinetics; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D054328:Proton Pump Inhibitors; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D009853:Omeprazole
keywords: Calorimetry; Conformational Destabilization; Crystal Structure; Cytochrome P450; Drug Metabolism; Enzyme Catalysis; Hydroxylase; Omeprazole; P450 BM3; Site-directed Mutagenesis