Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A reductive aminase from Aspergillus oryzae

Nature Chemistry volume 9, pages 961–969 (2017)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Jarvis, L. M. The year in new drugs. Chem. Eng. News 94, 12–17 (2016).

    Google Scholar 

  2. Topczewski, J. J., Cabrera, P. J., Saper, N. I. & Sanford, M. S. Palladium-catalysed transannular C–H functionalization of alicyclic amines. Nature 531, 220–224 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Mutti, F. G., Knaus, T., Scrutton, N. S., Breuer, M. & Turner, N. J. Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades. Science 349, 1525–1529 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Wanner, B., Kreituss, I., Gutierrez, O., Kozlowski, M. C. & Bode, J. W. Catalytic kinetic resolution of disubstituted piperidines by enantioselective acylation: synthetic utility and mechanistic insights. J. Am. Chem. Soc. 137, 11491–11497 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Xu, H. C., Chowdhury, S. & Ellman, J. A. Asymmetric synthesis of amines using tert-butanesulfinamide. Nat. Protoc. 8, 2271–2280 (2013).

    CAS  PubMed  Google Scholar 

  6. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science. 329, 305–309 (2010).

    CAS  PubMed  Google Scholar 

  7. Huang, H., Liu, X., Zhou, L., Chang, M. & Zhang, X. Direct asymmetric reductive amination for the synthesis of chiral β-arylamines. Angew. Chem. Int. Ed. 55, 5309–5312 (2016).

    CAS  Google Scholar 

  8. Nugent, T. C. & El-Shazly, M. Chiral amine synthesis—recent developments and trends for enamide reduction, reductive amination, and imine reduction. Adv. Synth. Catal. 352, 753–819 (2010).

    CAS  Google Scholar 

  9. Li, C., Villa-Marcos, B. & Xiao, J. Metal-bronsted acid cooperative catalysis for asymmetric reductive amination. J. Am. Chem. Soc. 131, 6967–6969 (2009).

    CAS  PubMed  Google Scholar 

  10. Schrittwieser, J. H., Velikogne, S. & Kroutil, W. Biocatalytic imine reduction and reductive amination of ketones. Adv. Synth. Catal. 357, 1655–1685 (2015).

    CAS  Google Scholar 

  11. Seiple, I. B. et al. A platform for the discovery of new macrolide antibiotics. Nature 533, 338–345 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Mathew, S. & Yun, H. ω-Transaminases for the production of optically pure amines and unnatural amino acids. ACS Catal. 2, 993–1001 (2012).

    CAS  Google Scholar 

  13. Simon, R. C., Richter, N., Busto, E. & Kroutil, W. Recent developments of cascade reactions involving ω-transaminases. ACS Catal. 4, 129–143 (2014).

    CAS  Google Scholar 

  14. Pavlidis, I. V. et al. Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines. Nat. Chem. 8, 1076–1082 (2016).

    CAS  PubMed  Google Scholar 

  15. Weise, N. J., Parmeggiani, F., Ahmed, S. T. & Turner, N. J. The bacterial ammonia lyase EncP: a tunable biocatalyst for the synthesis of unnatural amino acids. J. Am. Chem. Soc. 137, 12977–12983 (2015).

    CAS  PubMed  Google Scholar 

  16. DeLange, B. et al. Asymmetric synthesis of (S)-2-indolinecarboxylic acid by combining biocatalysis and homogeneous catalysis. ChemCatChem 3, 289–292 (2011).

    CAS  Google Scholar 

  17. Parmeggiani, F., Lovelock, S. L., Weise, N. J., Ahmed, S. T. & Turner, N. J. Synthesis of D- and L-phenylalanine derivatives by phenylalanine ammonia lyases: a multienzymatic cascade process. Angew. Chem. Int. Ed. 54, 4608–4611 (2015).

    CAS  Google Scholar 

  18. Ghislieri, D. et al. Engineering an enantioselective amine oxidase for the synthesis of pharmaceutical building blocks and alkaloid natural products. J. Am. Chem. Soc. 135, 10863–10869 (2013).

    CAS  PubMed  Google Scholar 

  19. Heath, R. S., Pontini, M., Bechi, B. & Turner, N. J. Development of an R-selective amine oxidase with broad substrate specificity and high enantioselectivity. ChemCatChem. 6, 996–1002 (2014).

    CAS  Google Scholar 

  20. Yasukawa, K., Nakano, S. & Asano, Y. Tailoring D-amino acid oxidase from the pig kidney to R-stereoselective amine oxidase and its use in the deracemization of α-methylbenzylamine. Angew. Chem. Int. Ed. 53, 4428–4431 (2014).

    CAS  Google Scholar 

  21. Chen, H. et al. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds. US patent 20130302859 (2013).

  22. Abrahamson, M. J., Vázquez-Figueroa, E., Woodall, N. B., Moore, J. C. & Bommarius, A. S. Development of an amine dehydrogenase for synthesis of chiral amines. Angew. Chem., Int. Ed. 51, 3969–3972 (2012).

    CAS  Google Scholar 

  23. Ye, L. J. et al. Engineering of amine dehydrogenase for asymmetric reductive amination of ketone by evolving Rhodococcus phenylalanine dehydrogenase. ACS Catal. 5, 1119–1122 (2015).

    CAS  Google Scholar 

  24. Mihara, H. et al. N-methyl-L-amino acid dehydrogenase from Pseudomonas putida: a novel member of an unusual NAD(P)-dependent oxidoreductase superfamily. FEBS J. 272, 1117–1123 (2005).

    CAS  PubMed  Google Scholar 

  25. Huber, T. et al. Direct reductive amination of ketones: structure and activity of S-selective imine reductases from Streptomyces. ChemCatChem. 6, 2248–2252 (2014).

    CAS  Google Scholar 

  26. Scheller, P. N., Lenz, M., Hammer, S. C., Hauer, B. & Nestl, B. M. Imine reductase-catalyzed intermolecular reductive amination of aldehydes and ketones. ChemCatChem. 7, 3239–3242 (2015).

    CAS  Google Scholar 

  27. Wetzl, D. et al. Asymmetric reductive amination of ketones catalyzed by imine reductases. ChemCatChem. 8, 2023–2026 (2016).

    CAS  Google Scholar 

  28. Mangas-Sanchez, J. et al. Imine reductases (IREDs). Curr. Opin. Chem. Biol. 37, 19–25 (2017).

    CAS  PubMed  Google Scholar 

  29. Leipold, F., Hussain, S., France, S. P. & Turner, N. J. in Science of Synthesis: Biocatalysis in Organic Synthesis 2 (eds Faber, K., Fessner, W.-D. & Turner, N. J.) 359–382 (Georg Thieme, 2015).

    Google Scholar 

  30. Leipold, F., Hussain, S., Ghislieri, D. & Turner, N. J. Asymmetric reduction of cyclic imines catalyzed by a whole-cell biocatalyst containing an (S)-imine reductase. ChemCatChem 5, 3505–3508 (2013).

    CAS  Google Scholar 

  31. Hussain, S. et al. An (R)-imine reductase biocatalyst for the asymmetric reduction of cyclic imines. ChemCatChem 7, 579–583 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Man, H. et al. Structure, activity and stereoselectivity of NADPH-dependent oxidoreductases catalysing the S-selective reduction of the imine substrate 2-methylpyrroline. ChemBioChem 16, 1052–1059 (2015).

    CAS  PubMed  Google Scholar 

  33. Rodriguez-Mata, M. et al. Structure and activity of NADPH-dependent reductase Q1EQE0 from Streptomyces kanamyceticus, which catalyses the R-selective reduction of an imine substrate. ChemBioChem 14, 1372–1379 (2013).

    CAS  PubMed  Google Scholar 

  34. Aleku, G. A. et al. Stereoselectivity and structural characterization of an imine reductase (IRED) from Amycolatopsis orientalis. ACS Catal. 6, 3880–3889 (2016).

    CAS  Google Scholar 

  35. Wetzl, D. et al. Expanding the imine reductase toolbox by exploring the bacterial protein-sequence space. ChemBioChem 16, 1749–1756 (2015).

    CAS  PubMed  Google Scholar 

  36. Scheller, P. N. et al. Enzyme toolbox: novel enantio-complimentary imine reductases. ChemBioChem 15, 2201–2204 (2014).

    CAS  PubMed  Google Scholar 

  37. Mitsukura, K., Suzuki, M., Tada, K., Yoshida, T. & Nagasawa, T. Asymmetric synthesis of chiral cyclic amine from cyclic imine by bacterial whole-cell catalyst of enantioselective imine reductase. Org. Biomol. Chem. 8, 4533–4535 (2010).

    CAS  PubMed  Google Scholar 

  38. Mitsukura, K. et al. Purification and characterization of a novel (R)-imine reductase from Streptomyces sp. GF3587. Biosci. Biotechnol. Biochem. 75, 1778–1782 (2011).

    CAS  PubMed  Google Scholar 

  39. Mitsukura, K. et al. A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression. Appl. Microbiol. Biotechnol. 97, 8079–8086 (2013).

    CAS  PubMed  Google Scholar 

  40. Fromm, H. G. in Initial Rate Enzyme Kinetics 121–160 (Springer, 1975).

  41. Fujioka, M. & Nakatani, Y. A kinetic study of saccharopine dehydrogenase reaction. Eur. J. Biochem. 16, 180–186 (1970).

    CAS  PubMed  Google Scholar 

  42. Heyde, E. & Ainsworth, S. Kinetic studies on the mechanism of the malate dehydrogenase reaction. J. Biol. Chem. 243, 2413–2423 (1968).

    CAS  PubMed  Google Scholar 

  43. Ohshima, T., Misono, H. & Soda, K. Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. J. Biol. Chem. 253, 5719–5725 (1978).

    CAS  PubMed  Google Scholar 

  44. Rife, J. E. & Cleland, W. W. Kinetic mechanism of glutamate dehydrogenase. Biochemistry 19, 2321–2328 (1980).

    CAS  PubMed  Google Scholar 

  45. Hochreiter, M. C., Patek, D. R. & Schellenberg, K. A. Catalysis of α-iminoglutarate formation from α-ketoglutarate and ammonia by bovine glutamate dehydrogenase. J. Biol. Chem. 247, 6271–6276 (1972).

    CAS  PubMed  Google Scholar 

  46. Stillman, T. J., Baker, P. J., Britton, K. L. & Rice, D. W. Conformational flexibility in glutamate dehydrogenase. role of water in substrate recognition and catalysis. J. Mol. Biol. 234, 1131–1139 (1993).

    CAS  PubMed  Google Scholar 

  47. Dairi, T. & Asano, Y. Cloning, nucleotide sequencing, and expression of an opine dehydrogenase gene from Arthrobacter sp. strain 1C. Appl Env. Microbiol. 61, 3169–3171 (1995).

    CAS  Google Scholar 

  48. Gand, M., Müller, H., Wardenga, R. & Höhne, M. Characterization of three novel enzymes with imine reductase activity. J. Mol. Catal. B 110, 126–132 (2014).

    CAS  Google Scholar 

  49. Rogers, T. A. & Bommarius, A. S. Utilizing simple biochemical measurements to predict lifetime output of biocatalysts in continuous isothermal processes. Chem. Eng. Sci. 65, 2118–2124 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Kohls, H., Steffen-Munsberg, F. & Höhne, M. Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis. Curr. Opin. Chem. Biol. 19, 180–192 (2014).

    CAS  PubMed  Google Scholar 

  51. Volner, A., Zoidakis, J. & Abu-Omar, M. M. Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases. J. Biol. Inorg. Chem. 8, 121–128 (2003).

    CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester, M1 7DN, UK

    Godwin A. Aleku, Scott P. France, Juan Mangas-Sanchez, Sarah L. Montgomery, Friedemann Leipold, Shahed Hussain & Nicholas J. Turner

  2. Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, YO10 5DD, York, UK

    Henry Man, Mahima Sharma & Gideon Grogan

Authors
  1. Godwin A. Aleku
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Scott P. France
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henry Man
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Juan Mangas-Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sarah L. Montgomery
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mahima Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Friedemann Leipold
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shahed Hussain
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gideon Grogan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Nicholas J. Turner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Gideon Grogan or Nicholas J. Turner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 8904 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2782

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing