Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine 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 establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l d.
Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field.
The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with e.e.s of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on 2 the age of the enzyme after purification. The structure of AoIRED has been determined in an 'open' apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N-and C-terminal domains of participating monomers. Co-crystallisation with NADPH gave a 'closed' form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by co-crystallization with NADPH and 1-methyl-1,2,3,4tetrahydroisoquinoline [(MTQ], and reveals a binding site for the (R)-amine product which places the chiral carbon within 4 Å of the putative location of the C4 atom of NADPH that delivers hydride to the C=N bond of the substrate. The ternary complex has permitted structureinformed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A, of altered activity and stereoselectivity.
Although the range of biocatalysts available for the synthesis of enantiomerically pure chiral amines continues to expand, few existing methods provide access to secondary amines. To address this shortcoming, we have over‐expressed the gene for an (R)‐imine reductase [(R)‐IRED] from Streptomyces sp. GF3587 in Escherichia coli to create a recombinant whole‐cell biocatalyst for the asymmetric reduction of prochiral imines. The (R)‐IRED was screened against a panel of cyclic imines and two iminium ions and was shown to possess high catalytic activity and enantioselectivity. Preparative‐scale synthesis of the alkaloid (R)‐coniine (90 % yield; 99 % ee) from the imine precursor was performed on a gram‐scale. A homology model of the enzyme active site, based on the structure of a closely related (R)‐IRED from Streptomyces kanamyceticus, was constructed and used to identify potential amino acids as targets for mutagenesis.
Oxidoreductases from Streptomyces sp. GF3546 [3546-IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2-methylpyrroline (2MPN) to (S)-2-methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue-Tyr169-in 3546-IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.
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