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Multifunctional biocatalyst for conjugate reduction and reductive amination

Nature volume 604, pages 86–91 (2022)Cite this article

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Abstract

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 (https://data.mendeley.com) at https://doi.org/10.17632/fhc429t33c.1. 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.

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Acknowledgements

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

Author notes

  1. Fabio Parmeggiani

    Present address: Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano, Milan, Italy

Authors and Affiliations

  1. Department of Chemistry, University of Manchester, Manchester Institute of Biotechnology, Manchester, UK

    Thomas W. Thorpe, James R. Marshall, Vanessa Harawa, Rebecca E. Ruscoe, Antonio Angelastro, Rachel S. Heath, Fabio Parmeggiani & Nicholas J. Turner

  2. Department of Chemistry, University of York, York, UK

    Anibal Cuetos & Gideon Grogan

  3. Prozomix, Haltwhistle, UK

    James D. Finnigan & Simon J. Charnock

  4. Pfizer Worldwide Research and Development, Groton, CT, USA

    Roger M. Howard & Rajesh Kumar

  5. Pfizer Worldwide Research and Development, Sandwich, UK

    David S. B. Daniels

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Contributions

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.

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Correspondence to Nicholas J. Turner.

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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). https://doi.org/10.1038/s41586-022-04458-x

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