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.
Highlights
The intelligent use of enzymes for the production of high-value fine chemicals, pharmaceuticals, and agrochemical intermediates is at the forefront of the drive toward the use of mild and ecologically sustainable manufacturing techniques.[1]
Recent work has demonstrated the biocatalytic potential of the Old Yellow Enzyme
Containing, NAD(P)H-dependent oxidoreductases are an exceptionally versatile group of biocatalysts and offer considerable potential for exploitation in industrial biocatalysis; the substrate specificity of these enzymes is generally confined to small molecules, which limits the scope of their applicability
Summary
The intelligent use of enzymes for the production of high-value fine chemicals, pharmaceuticals, and agrochemical intermediates is at the forefront of the drive toward the use of mild and ecologically sustainable manufacturing techniques.[1]. We determined the crystal structure of NtDBR both in isolation (holo) and in complexes with NADP(H) (binary) ± alkene substrate 4hydroxy-3-methoxycinnamaldehyde (HMCA) (ternary).
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