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Multifunctional biocatalyst for conjugate reduction and reductive amination


Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.

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Fig. 1: Enantioenriched amine diastereomers and one-pot enzymatic strategies for their synthesis.
Fig. 2: Substrate scope of EneIRED-catalysed CR–RA.
Fig. 3: Mechanistic and structural studies.
Fig. 4: Proposed catalytic cycle of productive EneIRED CR–RA and extension to six-electron CR–RA.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information, and NMR traces are available from the Mendeley data repository ( at Sequence data have been deposited in Genbank (accession numbers MW854365, MW925135–MW925140) and the coordinate files and structure factors have been deposited in the PDB with accession number 7A3W.


  1. Jarvis, L. M. The new drugs of 2019. Chem. Eng. News 98, 30–36 (2020).

    Google Scholar 

  2. Afanasyev, O. I., Kuchuk, E., Usanov, D. L. & Chusov, D. Reductive amination in the synthesis of pharmaceuticals. Chem. Rev. 119, 11857–11911 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Marshall, J. R. et al. Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination. Nat. Chem. 13, 140–148 (2021).

    Article  CAS  PubMed  Google Scholar 

  4. Roughley, S. D. & Jordan, A. M. The medicinal chemist’s toolbox: an analysis of reactions used in the pursuit of drug candidates. J. Med. Chem. 54, 3451–3479 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Yasukawa, T., Masuda, R. & Kobayashi, S. Development of heterogeneous catalyst systems for the continuous synthesis of chiral amines via asymmetric hydrogenation. Nat. Catal. 2, 1088–1092 (2019).

    Article  CAS  Google Scholar 

  6. Wu, Z. et al. Secondary amines as coupling partners in direct catalytic asymmetric reductive amination. Chem. Sci. 10, 4509–4514 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Skrypai, V., Varjosaari, S. E., Azam, F., Gilbert, T. M. & Adler, M. J. Chiral Brønsted acid-catalyzed metal-free asymmetric direct reductive amination using 1-hydrosilatrane. J. Org. Chem. 84, 5021–5026 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Aleku, G. A. et al. A reductive aminase from Aspergillus oryzae. Nat. Chem. 9, 961–969 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Mayol, O. et al. A family of native amine dehydrogenases for the asymmetric reductive amination of ketones. Nat. Catal. 2, 324–333 (2019).

    Article  CAS  Google Scholar 

  10. Yang, Y., Cho, I., Qi, X., Liu, P. & Arnold, F. H. An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)–H bonds. Nat. Chem. 11, 987–993 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li, T. et al. Efficient, chemoenzymatic process for, anufacture of the Boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization.J. Am. Chem. Soc. 134, 6467–6472 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Zhou, J. & List, B. Organocatalytic asymmetric reaction cascade to substituted cyclohexylamines. J. Am. Chem. Soc. 129, 7498–7499 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Monti, D. et al. Cascade coupling of ene-reductases and ω-transaminases for the stereoselective synthesis of diastereomerically enriched amines. ChemCatChem 7, 3106–3109 (2015).

    Article  CAS  Google Scholar 

  14. France, S. P., Hepworth, L. J., Turner, N. J. & Flitsch, S. L. Constructing biocatalytic cascades: in vitro and in vivo approaches to de novo multi-enzyme pathways. ACS Catal. 7, 710–724 (2017).

    Article  CAS  Google Scholar 

  15. Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Toogood, H. S. & Scrutton, N. S. Discovery, Characterization, engineering, and applications of ene-reductases for industrial biocatalysis. ACS Catal. 8, 3532–3549 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Roth, S., Kilgore, M. B., Kutchan, T. M. & Müller, M. Exploiting the catalytic diversity of short-chain dehydrogenases/reductases: versatile enzymes from plants with extended imine substrate scope. ChemBioChem 19, 1849–1852 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Hyslop, J. F. et al. Biocatalytic synthesis of chiral N-functionalized amino acids. Angew. Chemie. Int. Ed. 57, 13821–13824 (2018).

    Article  CAS  Google Scholar 

  19. Kato, Y., Yamada, H. & Asano, Y. Stereoselective synthesis of opine-type secondary amine carboxylic acids by a new enzyme opine dehydrogenase use of recombinant enzymes. J. Mol. Catal. B 1, 151–160 (1996).

    Article  CAS  Google Scholar 

  20. Schober, M. et al. Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase. Nat. Catal. 2, 909–915 (2019).

    Article  CAS  Google Scholar 

  21. Bornadel, A. et al. Technical considerations for scale-up of imine-reductase-catalyzed reductive amination: a case study. Org. Process Res. Dev. 23, 1262–1268 (2019).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yao, P., Xu, Z., Yu, S., Wu, Q. & Zhu, D. Imine reductase-catalyzed enantioselective reduction of bulky α,β-unsaturated imines en route to a pharmaceutically important morphinan skeleton. Adv. Synth. Catal. 361, 556–561 (2019).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. Lenz, M. et al. Asymmetric ketone reduction by imine reductases. ChemBioChem 18, 253–256 (2017).

    Article  CAS  PubMed  Google Scholar 

  26. Thorpe, T. W. et al. One-pot biocatalytic cascade reduction of cyclic enimines for the preparation of diastereomerically enriched N-heterocycles. J. Am. Chem. Soc. 141, 19208–19213 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Steiningerova, L. et al. Different reaction specificities of F420H2-dependent reductases facilitate pyrrolobenzodiazepines and lincomycin to fit their biological targets. J. Am. Chem. Soc. 142, 3440–3448 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Trenti, F. et al. Early and late steps of quinine biosynthesis. Org. Lett. 23, 1793–1797 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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).

    Article  CAS  PubMed  Google Scholar 

  30. France, S. P. et al. Identification of novel bacterial members of the imine reductase enzyme family that perform reductive amination. ChemCatChem 10, 510–514 (2018).

    Article  CAS  Google Scholar 

  31. Mangas-Sanchez, J. et al. Asymmetric synthesis of primary amines catalyzed by thermotolerant fungal reductive aminases. Chem. Sci. 11, 5052–5057 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Montgomery, S. L. et al. Characterization of imine reductases in reductive amination for the exploration of structure–activity relationships. Sci. Adv. 6, 9320 (2020).

    Article  ADS  CAS  Google Scholar 

  33. Ouellet, S. G., Walji, A. M. & Macmillan, D. W. C. Enantioselective organocatalytic transfer hydrogenation reactions using Hantzsch esters. Acc. Chem. Res. 40, 1327–1339 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Santi, N., Morrill, L. C., Świderek, K., Moliner, V. & Luk, L. Y. P. Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts. Chem. Commun. 57, 1919–1922 (2021).

    Article  CAS  Google Scholar 

  35. Rodríguez-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).

    Article  PubMed  CAS  Google Scholar 

  36. Holm, L. Benchmarking fold detection by DaliLite v.5. Bioinformatics 35, 5326–5327 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. Lenz, M. et al. New imine-reducing enzymes from β-hydroxyacid dehydrogenases by single amino acid substitutions. Protein Eng. Des. Sel. 31, 109–120 (2018).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2009).

    Google Scholar 

  42. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kabsch, W. XDS. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  44. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. Sect. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  CAS  Google Scholar 

  45. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  46. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  47. Sharma, M. et al. A mechanism for reductive amination catalyzed by fungal reductive aminases. ACS Catal. 8, 11534–11541 (2018).

    Article  CAS  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  49. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

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T.W.T. is grateful for a CASE award from the UK Biotechnology and Biological Sciences Research Council (BBSRC) and Pfizer (BB/M011208/1). J.R.M. acknowledges a CASE award from the Industrial Biotechnology Innovation Centre (IBioIC), BBSRC and Prozomix Ltd. A.C. was funded by grant BB/P005578/1 from the BBSRC. N.J.T. is grateful to the ERC for the award of an Advanced Grant (742987). We thank J. P. Turkenburg and S. Hart for assistance with X-ray data collection and the Diamond Light Source for access to beamline I03 under proposal number mx-9948.

Author information

Authors and Affiliations



N.J.T., G.G., R.M.H., R.K. and D.S.B.D. devised and supervised the project. F.P. and R.E.R. managed the project. T.W.T. and A.A. performed mechanistic studies. T.W.T. and R.E.R. carried out substrate scope reactions. T.W.T. and V.H. carried out preparative scale reactions. T.W.T., R.E.R and V.H. synthesized substrates and standards. A.C., T.W.T. and G.G. performed crystallographic and docking studies. T.W.T. and R.S.H. undertook site-directed mutagenesis. J.R.M., S.J.C. and J.D.F. performed genetic identification, cloning and bioinformatics. T.W.T. and J.R.M. produced and purified the biocatalyst. N.J.T., G.G., R.M.H., R.K., D.S.B.D., S.J.C., F.P., J.D.F., A.C., R.E.R., V.H., J.R.M. and T.W.T. wrote the manuscript and generated the figures.

Corresponding author

Correspondence to Nicholas J. Turner.

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The authors declare no competing interests.

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Nature thanks Dominic Campopiano, Sandy Schmidt and Thomas Ward for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Phylogenetic IRED tree mapped against the reaction profiles of IRED-catalysed reduction of ene-imine I.

Although the majority of the IREDs catalysed conventional imine reduction only, a small number were able to reduce both C=C and C=N bonds. Of these, EneIRED (pIR-120) possessed the highest propensity to forming the desired amine II.

Extended Data Fig. 2 Optimization of EneIRED-catalysed CR–RA reaction conditions.

Conversion to the products of CR and CR–RA were elevated in glycine-OH pH 9.0, at moderate DMSO cosolvent concentration and at higher equivalents of amine donor. Formation of the direct RA product was not observed under any conditions.

Extended Data Fig. 3 Scaled-up examples of EneIRED-catalysed CR–RA.

Several secondary and tertiary amines could be prepared including an example at elevated enone concentration and lower amine equivalents.

Extended Data Fig. 4 Control reactions and isolated reactions of potential CR–RA pathway intermediates in EneIRED-catalysed CR–RA.

a, EneIRED CR–RA of 15 and b with NADPH. b, No enzyme control reaction. c, No recycling system control reaction. d, Reactions of potential CR–RA intermediate 15b with NADP+ or NADPH. e, Reaction of potential CR–RA intermediate 15′ with b using EneIRED. f, No amine control reactions with EneIRED point variants.

Extended Data Fig. 5 Time-course studies of the CR–RA of 15 with b catalysed by wild-type EneIRED and point variants.

Both EneIRED-Y177A and EneIRED-Y181A exhibited a reduction in the rate of CR and CR–RA product formation compared to wild-type EneIRED, indicating that both residues are important for efficient catalysis. Notably, for EneIRED-Y177A the concentration of the ketone intermediate was comparatively low throughout the reaction, suggesting that Y177 is more important for CR than RA.

Extended Data Fig. 6 Active site of EneIRED highlighting electron density.

a, Side chains, with density corresponding to the refined 2Fo − Fc map (blue) at a level of 1σ. b, NADP+, with density corresponding to the Fo − Fc difference map (green) at a level of 3σ obtained from refinement in the absence of the ligand, with refined atoms included for clarity. Fo and Fc stand for the observed and calculated structure factor amplitudes, respectively.

Extended Data Table 1 Data collection and refinement statistics (molecular replacement) for EneIRED in complex with NADP+

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Thorpe, T.W., Marshall, J.R., Harawa, V. et al. Multifunctional biocatalyst for conjugate reduction and reductive amination. Nature 604, 86–91 (2022).

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