Preissler, JaninaReeve, Holly A.Zhu, TianzeNicholson, JakeUrata, KoujiLauterbach, LarsWong, Luet L.Vincent, Kylie A.Lenz, Oliver2021-06-102021-06-102020-06-161867-3880https://depositonce.tu-berlin.de/handle/11303/13228http://dx.doi.org/10.14279/depositonce-12023The 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.en540 Chemie und zugeordnete Wissenschaftenhydrogenasemetalloenzymecytochrome P450monooxygenaseoxidoreductaseimine reductaseoctane oxidationnicotinamide cofactorNADHcofactor recyclingArticle1867-3899