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A family of native amine dehydrogenases for the asymmetric reductive amination of ketones

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Abstract

The asymmetric reductive amination of ketones enables the one-step synthesis of chiral amines from readily available starting materials. Here we report the discovery of a family of native NAD(P)H-dependent amine dehydrogenases (nat-AmDHs) competent for the asymmetric reductive amination of aliphatic and alicyclic ketones, adding significantly to the biocatalytic toolbox available for chiral amine synthesis. Studies of ketone and amine substrate specificity and kinetics reveal a strong preference for aliphatic ketones and aldehydes, with activities of up to 614.5 mU mg−1 for cyclohexanone with ammonia, and 851.3 mU mg−1 for isobutyraldehyde with methylamine as the amine donor. Crystal structures of three nat-AmDHs (AmDH4, MsmeAmDH and CfusAmDH) reveal the active site determinants of substrate and cofactor specificity and enable the rational engineering of AmDH4 for the generated activity towards pentan-2-one. Analysis of the three-dimensional catalytic site distribution among bacterial biodiversity revealed a superfamily of divergent proteins with representative specificities ranging from amino acid substrates to hydrophobic ketones.

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Fig. 1: Amine dehydrogenases identified in this study.
Fig. 2: SSN of the native AmDH family.
Fig. 3: Structural and mutagenesis data of AmDH4.
Fig. 4: Structure of the active site of CfusAmDH and MsmeAmDH.
Fig. 5: Dividing the AmDH family into groups with similar active sites.

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Data availability

Crystallographic data that support the findings of this study have been deposited in the Protein Data Bank (PDB) under accession codes 6G1H, 6G1M, 6IAU and 6IAQ. All the other data supporting the findings of this study are available within the paper and its Supplementary Information and Supplementary Data or from the corresponding authors upon reasonable request.

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Acknowledgements

The authors thank M. Salanoubat for supporting the project, N. Fonknechten for fruitful discussions and assistance with genomic context analysis, C. Pelle and P. Sirvain for large-scale purification and analytical gel filtration of the described enzymes, O. Maciejak (University Val d’Essonne) for NMR assistance, the Region Ile de France for financial support of the 600 MHz spectrometer, S. Hart and J Agirre for assistance with X-ray data collection and data analysis respectively, and the Diamond Light Source for access to beamlines I04 and I04-1 under proposal no. mx-9948. This work was supported by Commissariat à l’énergie atomique et aux énergies alternatives (CEA), the CNRS and the University of Evry Val d’Essonne. The authors thank GlaxoSmithKline for the award of a part studentship to A.F., the Brazilian Government for a fellowship to L.B. under the Coordination for the Improvement of Higher Education Personnel (CAPES) scheme and the COST Action CM1303 ‘System Biocatalysis’ for STSM of O.M in G.G.’s laboratory.

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Authors and Affiliations

  1. Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057, EVRY, France

    Ombeline Mayol, Karine Bastard, Jean-Louis Petit, Aline Mariage, Adrien Debard, Virginie Pellouin, Alain Perret, Véronique de Berardinis, Anne Zaparucha & Carine Vergne-Vaxelaire

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

    Lilian Beloti, Amina Frese, Johan P. Turkenburg & Gideon Grogan

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  1. Ombeline Mayol
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Contributions

C.V.V. conceived the project and directed it with G.G. C.V.V., G.G., O.M., A.Z. and V.d.B. supervised the project. V.d.B. and J.-L.P. performed the candidate enzyme selection. A.D., V.P. carried out the gene cloning, the protein expression and purification on a small scale and the enzymatic screening with input from A.M. and J.-L.P.. O.M., V.P. and C.V.V. conducted the specific activity measurements. G.G., J.P.T., A.F. and L.B. conducted all the structural resolution. K.B. conceived and conducted the structural bioinformatics analysis with input from O.M., C.V.V. and G.G. O.M. carried out the biochemical experiments under the supervision of A.P. O.M. and C.V.V. performed the analytical and semi-preparative scale reactions. C.V.V., G.G., K.B. and O.M. wrote the manuscript with input from A.P., A.Z. and V.d.B.

Corresponding authors

Correspondence to Gideon Grogan or Carine Vergne-Vaxelaire.

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Supplementary information

Supplementary Information

Supplementary Figures 1–32, Supplementary Tables 1–9, Supplementary Methods, Supplementary Note 1, Supplementary Discussion, Supplementary References

Reporting Summary

Supplementary Data 1

model of AmDH4 (closed form) in complex with NAD+, and product 2,4-DAP

Supplementary Data 2

model of AmDH4 (closed form) in complex with NAD+, ammonia and substrate (2R)−2A4OP

Supplementary Data 3

model of AmDH4 variant N135V/N163V/R161M/H264L in complex with NAD+, ammonia and pentan-2-one

Supplementary Data 4

model of CfusAmDH in complex with NADP+, ammonia and cyclohexanone

Supplementary Data 5

model of MsmeAmDH in complex with NADP+, ammonia and cyclohexanone

Supplementary Data 6

list of the 5313 proteins (Uniprot ID) used in the Sequence Similarity Network

Supplementary Data 7

multiple alignment on the whole sequences of proteins from G3 and G4 groups

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Mayol, O., Bastard, K., Beloti, L. et al. A family of native amine dehydrogenases for the asymmetric reductive amination of ketones. Nat Catal 2, 324–333 (2019). https://doi.org/10.1038/s41929-019-0249-z

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