Biocatalytic AsymmetricAlkene Reduction: CrystalStructure and Characterization of a Double Bond Reductase from Nicotiana tabacum

摘要

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 exocyclicC=C double bond of (R)-pulegone) and in somecases showing an opposite stereopreference in comparison with theOYE family member pentaerythritol tetranitrate (PETN) reductase. Thisserves to augment classical OYE “-ene” reductase activityand, coupled with its aerobic stability, emphasizes the potentialindustrial value of NtDBR. Furthermore, we also report the X-ray crystalstructures 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-saturatedmutagenesis studies aimed at enhancing the reactivity, stereochemistry,and specificity of this enzyme.