Dihydrogen-Driven NADPH Recycling in Imine Reduction and P450-Catalyzed Oxidations Mediated by an Engineered O2-Tolerant Hydrogenase

The O-2-tolerant NAD(+)-reducing hydrogenase (SH) fromRalstonia eutropha(Cupriavidus necator) has already been appliedin vitroandin vivofor H-2-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 O-2-tolerance. The applicability of the SH variant in H-2-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 H-2/O(2)mixtures. Thus, the re-designed SH represents a versatile platform for atom-efficient, H-2-driven cofactor recycling in biotransformations involving NADPH-dependent oxidoreductases.