title: Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.
abstract: Three enzymes of theMenthaessential 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: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: Antonios
authors: A
authors: Lygidakis
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: Vijaykumar
authors: V
authors: Karuppiah
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: Robin
authors: 0000-0002-2825-3320
authors: R
authors: Hoeven
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: Aisling
authors: A
authors: Ní Cheallaigh
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: David
authors: 0000-0003-4845-8443
authors: D
authors: Leys
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: John M
authors: 0000-0003-1827-6817
authors: JM
authors: Gardiner
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: Helen S
authors: 0000-0003-4797-0293
authors: HS
authors: Toogood
authors: Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.
authors: Nigel S
authors: 0000-0002-4182-3500
authors: NS
authors: Scrutton
keywords: Biosynthese essenzieller Mentha-Öle; Biotransformationen; Isopiperitenon-Reduktase; Kurzkettige Dehydrogenasen/Reduktasen; Strukturaufklärung