Dihydrogen‐Driven NADPH Recycling in Imine Reduction and P450‐Catalyzed Oxidations Mediated by an Engineered O 2 ‐Tolerant Hydrogenase

The O 2 ‐tolerant NAD + ‐reducing hydrogenase (SH) from Ralstonia eutropha ( Cupriavidus necator ) has already been applied in vitro and in vivo for 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.