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Development of imine reductases and reductive aminases for chiral amine synthesis

Reductive amination is one of the most important methods for the synthesis of chiral amines. Herein, we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) which can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines in up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine substrate show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies of AspRedAm suggest a mechanism involving ordered binding of substrates and release of products, consistent with an enzyme capable of catalysing imine formation as well as reduction. The crystal structures of AspRedAm in complex with NADP(H) and with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline have been obtained and used to inform site-directed mutagenesis studies, which have highlighted possible catalytic roles for D169 and Y177, and substrate recognition roles for Q240 and W210. The AspRedAm wild-type and Q240A variant biocatalysts have been applied to reductive amination reactions on a preparative scale, displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g L d.