Discovery and synthetic applications of a NAD(P)Hdependent reductive aminase from Rhodococcus erythropolis

Reductive amination is one of the most synthetically direct routes to access chiral amines. Several Imine Reductases (IREDs) have been discovered to catalyze reductive amination (Reductive Aminases or RedAms), yet are dependent on the expensive phosphorylated nicotinamide adenine dinucleotide cofactor NADPH and usually more active at basic pH. Here we describe the discovery and synthetic potential of an IRED from Rhodococcus erythropolis (RytRedAm) that catalyzes reductive amination between a series of medium to large carbonyl and amine compounds with conversions of up to >99% and 99% enantiomeric excess at neutral pH. RytRedAm catalyzes the formation of a substituted γ- lactam and N-methyl-1-phenylethanamine with opposite stereochemistry to that of fungal RedAms, giving the (S)-enantiomer. This enzyme remarkably uses both NADPH and NADH cofactors with KM values of 15 and 247 μM, and turnover numbers kcat of 3.6 and 9.0 s-1, respectively, for the reductive amination of hexanal with allylamine. The crystal structure obtained provides insights in the flexibility to also accept NADH, with residues R35 and I69 diverging from that of other IREDs/RedAms in the otherwise conserved Rossmann fold. RytRedAm thus represents a subfamily of enzymes that enable synthetic applications using NADH-dependent reductive amination to access complementary chiral amine products.