title: Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase fromNicotiana tabacum.
abstract: The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase fromNicotiana tabacum(NtDBR), which belongs to the leukotriene B4dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: David J
authors.initials: DJ
authors.lastname: Mansell
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: Helen S
authors.initials: HS
authors.lastname: Toogood
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: John
authors.initials: J
authors.lastname: Waller
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: John M X
authors.initials: JM
authors.lastname: Hughes
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: Colin W
authors.initials: CW
authors.lastname: Levy
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: John M
authors.initials: JM
authors.lastname: Gardiner
authors.affiliation: Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
keywords: Nicotiana tabacum; asymmetric alkene reduction; biocatalysis; crystal structure; double bond reductase