Abstract
AbstractThe O2‐tolerant NAD+‐reducing hydrogenase (SH) from Ralstonia eutropha (Cupriavidus necator) has already been applied in vitro and in vivo for H2‐driven NADH recycling in coupled enzymatic reactions with various NADH‐dependent oxidoreductases. To expand the scope for application in NADPH‐dependent biocatalysis, we introduced changes in the NAD+‐binding pocket of the enzyme by rational mutagenesis, and generated a variant with significantly higher affinity for NADP+ than for the natural substrate NAD+, while retaining native O2‐tolerance. The applicability of the SH variant in H2‐driven NADPH supply was demonstrated by the full conversion of 2‐methyl‐1‐pyrroline into a single enantiomer of 2‐methylpyrrolidine catalysed by a stereoselective imine reductase. In an even more challenging reaction, the SH supported a cytochrome P450 monooxygenase for the oxidation of octane under safe H2/O2 mixtures. Thus, the re‐designed SH represents a versatile platform for atom‐efficient, H2‐driven cofactor recycling in biotransformations involving NADPH‐dependent oxidoreductases.
Highlights
One of the most commonly used cofactor recycling systems for NAD(P)H is glucose-(6P) dehydrogenase which reduces NAD (P) + at the expense of glucose-(6P) oxidation.[4]
Sustained H2-driven NADP + reduction in the presence of O2 has Biocatalysis is becoming established as a valuable tool in the production of complex chemicals.[1]
Altering substrate specificity of NAD + -reducing soluble hydrogenases (SHs) by rational design required the identification of amino acid residues that are involved in substrate binding
Summary
One of the most commonly used cofactor recycling systems for NAD(P)H is glucose-(6P) dehydrogenase which reduces NAD (P) + at the expense of glucose-(6P) oxidation.[4]. Sustained H2-driven NADP + reduction in the presence of O2 has Biocatalysis is becoming established as a valuable tool in the production of complex chemicals.[1] In particular biocatalytic transformations for asymmetric reductions including ketone, alkene and imine reductions, and for controlled oxidations such as terminal alcohol to aldehyde conversions and C H bond activations, are becoming accepted as promising alternatives to traditional chemical methods.[2] the enzymes that catalyze these transformations rely on electron transfer to or from redox equivalents which in most cases are the expensive biological cofactors NAD(P) + or NAD(P)H.[3] To develop viable biocatalytic processes, it is essential to have efficient systems for recycling these cofactors. The latter case represents the first example of H2-driven NADPH recycling to support an O2-dependent biocatalytic process
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