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Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short-Chain Dehydrogenases/Reductases
Authors:
Antonios
Lygidakis
(Manchester Institute of Biotechnology, University of Manchester)
,
Vijaykumar
Karuppiah
(Manchester Institute of Biotechnology, University of Manchester, Diamond Light Source)
,
Robin
Hoeven
(Manchester Institute of Biotechnology, University of Manchester)
,
Aisling
Ní Cheallaigh
(Manchester Institute of Biotechnology, University of Manchester)
,
David
Leys
(Manchester Institute of Biotechnology, University of Manchester, Diamond Light Source)
,
John M.
Gardiner
(Manchester Institute of Biotechnology, University of Manchester)
,
Helen S.
Toogood
(Manchester Institute of Biotechnology, University of Manchester)
,
Nigel S.
Scrutton
(Manchester Institute of Biotechnology, University of Manchester)
Co-authored by industrial partner:
No
Type:
Journal Paper
Journal:
Angewandte Chemie
, VOL 128
, PAGES 9748 - 9752
State:
Published (Approved)
Published:
August 2016
Diamond Proposal Number(s):
8997

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.
Subject Areas:
Biology and Bio-materials,
Chemistry,
Food Science
Instruments:
I02-Macromolecular Crystallography
,
I03-Macromolecular Crystallography
,
I04-1-Macromolecular Crystallography (fixed wavelength)