title: Enantioselective Benzylic Hydroxylation Catalysed by P450 Monooxygenases: Characterisation of a P450cam Mutant Library and Molecular Modelling.
abstract: Cytochrome P450 monooxygenases can catalyse the stereoselective C-H activation of a very broad range of substrates. Prediction and control of enantioselectivity of this enzyme class is of great interest for the synthesis of high-value chiral molecules. Here we have used a combination of molecular dynamics simulations and experimental screening to study the enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene. Small variations either in substrate structure or in enzyme active site architecture were shown to lead to dramatic changes in enantioselectivity; this was broadly in agreement with computational predictions. In addition to validating computational approaches, these studies have provided us with a deeper understanding of effects that might control stereoselectivity in these biooxidation reactions.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
authors.forename: Anja
authors.initials: A
authors.lastname: Eichler
authors.affiliation: Institute for Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Gricman
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
authors.forename: Susanne
authors.initials: S
authors.lastname: Herter
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Kelly
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Turner
authors.affiliation: Institute for Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. juergen.pleiss@itb.uni-stuttgart.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Pleiss
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. sabine.flitsch@manchester.ac.uk.
authors.forename: Sabine L
authors.identifier: http://orcid.org/0000-0003-3974-646X
authors.initials: SL
authors.lastname: Flitsch
mesh_terms: D001593:Benzyl Compounds / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D008958:Models, Molecular; D009154:Mutation; D013237:Stereoisomerism
chemical_list: D001593:Benzyl Compounds; D003577:Cytochrome P-450 Enzyme System
keywords: C−H activation; P450 enzymes; benzylic oxidation; chiral alcohols; directed evolution; enantioselectivity