title: Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.
abstract: Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: Antonios
authors.initials: A
authors.lastname: Lygidakis
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: Vijaykumar
authors.initials: V
authors.lastname: Karuppiah
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: Robin
authors.identifier: 0000-0002-2825-3320
authors.initials: R
authors.lastname: Hoeven
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: Aisling
authors.initials: A
authors.lastname: Ní Cheallaigh
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: David
authors.identifier: 0000-0003-4845-8443
authors.initials: D
authors.lastname: Leys
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: John M
authors.identifier: 0000-0003-1827-6817
authors.initials: JM
authors.lastname: Gardiner
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
authors.forename: Helen S
authors.identifier: 0000-0003-4797-0293
authors.initials: HS
authors.lastname: Toogood
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK. nigel.scrutton@manchester.ac.uk.
authors.forename: Nigel S
authors.identifier: 0000-0002-4182-3500
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D015394:Molecular Structure; D009822:Oils, Volatile / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: D009822:Oils, Volatile; D010088:Oxidoreductases
keywords: Mentha essential oil biosynthesis; biotransformations; isopiperitenone reductase; short-chain dehydrogenases/reductases; structure elucidation