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 (NtDBR)， which belongs to the leukotriene B dehydrogenase (LTD) subfamily of the zinc-independent， medium chain dehydrogenase/reductase superfamil... More
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 (NtDBR)， which belongs to the leukotriene B 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 ()-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 ()-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity， stereochemistry， and specificity of this enzyme.
