Biocatalytic asymmetric alkene reduction: Crystal structure and characterization of a double bond reductase from Nicotiana tabacum

David J. Mansell, Helen S. Toogood, John Waller, John M X Hughes, Colin W. Levy, John M. Gardiner, Nigel S. Scrutton

Research output: Contribution to journalArticlepeer-review

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 from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (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. © 2013 American Chemical Society.
Original languageEnglish
Pages (from-to)370-379
Number of pages9
JournalACS Catalysis
Volume3
Issue number3
DOIs
Publication statusPublished - 2013

Keywords

  • asymmetric alkene reduction
  • biocatalysis
  • crystal structure
  • double bond reductase
  • Nicotiana tabacum

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology

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