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Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short-Chain Dehydrogenases/Reductases

DOI: 10.1002/ange.201603785 DOI Help

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

Open Access Open Access

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)