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A reductive aminase from Aspergillus oryzae

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Abstract

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−1 d−1.

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Figure 1: Examples of biocatalytic routes to chiral amines via monoamine oxidase catalysed resolution, or asymmetric synthesis catalysed by ammonia lyases, transaminases, amine dehydrogenases and imine reductases (IREDs).
Figure 2: Reactivity chart for AspRedAm-catalysed reactions based on specific activities of a panel of carbonyl compounds and amine reacting partners.
Figure 3: Reductive amination of 1 with g and kinetic model for AspRedAm showing sequential cofactor and substrate binding followed by product and cofactor release based on steady-state kinetic studies.
Figure 4: Structural and mutagenesis data of AspRedAm highlighting essential catalytic residues.

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Acknowledgements

We thank the industrial affiliates of the Centre of Excellence for Biocatalysis, Biotransformations and Biomanufacture (CoEBio3) for awarding studentships to G.A.A. and H.M.. S.P.F. was supported by a CASE studentship from Pfizer. J.M.S and M.S. were funded by grant BB/M006832/1 from the UK Biotechnology and Biological Sciences Research Council. S.L.M. was supported by a CASE studentship from Johnson Matthey. S.H. was supported by a CASE studentship from AstraZeneca. F.L. received support from the Innovative Medicines Initiative Joint Undertaking under the grant agreement no. 115360 (Chemical manufacturing methods for the 21st century pharmaceutical industries, CHEM21) and the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in-kind contributions. We thank J. P. Turkenburg and S. Hart for assistance with X-ray data collection, and the Diamond Light Source for access to beamlines I02 and I03 under proposal number mx-9948. The authors would also like to thank J. Citoler for assistance with mutagenesis. N.J.T. also acknowledges the Royal Society for a Wolfson Research Merit Award.

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N.J.T. and G.G. initiated the study and directed the project. G.A.A., M.S. and F.L. cloned and expressed the enzymes. G.A.A. performed the kinetics and mutagenesis studies. G.A.A., S.P.F., J.M.-S., S.L.M. and M.S. performed biotransformations. H.M. obtained crystal structures. S.P.F., J.M.-S., S.L.M., G.A.A. and S.H. chemically synthesized substrates and product standards.

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Correspondence to Gideon Grogan or Nicholas J. Turner.

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Aleku, G., France, S., Man, H. et al. A reductive aminase from Aspergillus oryzae. Nature Chem 9, 961–969 (2017). https://doi.org/10.1038/nchem.2782

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