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ABSTRACT Chiral amines can be made by insertion of a carbene into an N–H bond using two-catalyst systems that combine a transition metal-based carbene-transfer catalyst and a chiral

proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N–H insertion, but asymmetric protonation in an active site replete with proton sources is challenging.

Here we describe engineered cytochrome P450 enzymes that catalyse carbene N–H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to

32,100 total turnovers, >99% yield and 98% e.e.). These enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent

stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this

transformation and provide stereocontrol. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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support SIMILAR CONTENT BEING VIEWED BY OTHERS BIOCATALYTIC, ENANTIOENRICHED PRIMARY AMINATION OF TERTIARY C–H BONDS Article 03 May 2024 CHEMODIVERGENT C(_SP_3)–H AND C(_SP_2)–H

CYANOMETHYLATION USING ENGINEERED CARBENE TRANSFERASES Article 19 January 2023 MULTIFUNCTIONAL BIOCATALYST FOR CONJUGATE REDUCTION AND REDUCTIVE AMINATION Article 06 April 2022 DATA

AVAILABILITY All data necessary to support the paper’s conclusions are available in the main text and the Supplementary Information. X-ray crystal structures of 3E (CCDC 2065484) and 3L

(CCDC 2065489) are available free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Plasmids encoding the enzymes reported in this study are

available for research purposes from F.H.A. under a material transfer agreement with the California Institute of Technology. Source data are provided with this paper. REFERENCES * Hili, R.

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62, 872–881 (1979). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the National Science Foundation Division of Molecular and Cellular

Biosciences (grant 2016137 to F.H.A.), the US Army Research Office Institute for Collaborative Biotechnologies (cooperative agreement W911NF-19-2-0026 to F.H.A.), the Spanish Ministry of

Science and Innovation MICINN (grant PID2019-111300GA-I00 to M.G.-B.) and the Generalitat de Catalunya AGAUR Beatriu de Pinós H2020 MSCA-Cofund (2018-BP-00204 project to M.G.-B.). K.C.

thanks the Resnick Sustainability Institute at Caltech for fellowship support. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged

(RES-QSB-2020-2-0016). We thank D. C. Miller, S. Brinkmann-Chen, R. Lal and T. Zeng for helpful discussions and comments on the manuscript. We further thank M. Shahgholi for high-resolution

mass spectrometry analysis and M. K. Takase for X-ray crystallographic analysis. AUTHOR INFORMATION Author notes * Kai Chen Present address: Innovative Genomics Institute, University of

California, Berkeley, CA, USA AUTHORS AND AFFILIATIONS * Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA Zhen Liu, Andrew Z. Zhou, Kai

Chen & Frances H. Arnold * Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, Girona, Spain Carla Calvó-Tusell & Marc Garcia-Borràs

Authors * Zhen Liu View author publications You can also search for this author inPubMed Google Scholar * Carla Calvó-Tusell View author publications You can also search for this author

inPubMed Google Scholar * Andrew Z. Zhou View author publications You can also search for this author inPubMed Google Scholar * Kai Chen View author publications You can also search for this

author inPubMed Google Scholar * Marc Garcia-Borràs View author publications You can also search for this author inPubMed Google Scholar * Frances H. Arnold View author publications You can

also search for this author inPubMed Google Scholar CONTRIBUTIONS Z.L. and K.C. conceived and designed the overall project with F.H.A. providing guidance. Z.L. and A.Z.Z. designed and

performed the initial screening of haem proteins and the substrate scope study. C.C.-T. and M.G.-B. carried out the computational studies. Z.L., K.C., M.G.-B. and F.H.A. wrote the manuscript

with the input of all authors. CORRESPONDING AUTHORS Correspondence to Kai Chen, Marc Garcia-Borràs or Frances H. Arnold. ETHICS DECLARATIONS COMPETING INTERESTS K.C., Z.L. and A.Z.Z. are

inventors on a US Patent Application (invention title, Diverse Carbene Transferase Enzyme Catalysts Derived from a P450 Enzyme; application no., 17/200,394) filed by the California Institute

of Technology, which covers lactone-carbene N–H insertion with engineered P450 enzymes. The patent was filed on 12 March 2021. The remaining authors declare no competing interests.

ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Chemistry_ thanks Sabine Flitsch, Sason Shaik and the other, anonymous, reviewer(s) for their contribution to the peer review of this

work. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY

INFORMATION Supplementary Figs. 1–21, Tables 1–17, experimental data, procedural details, synthesis and characterization data, NMR spectra, X-ray crystallographic data and computational

modelling data. SUPPLEMENTARY DATA 1 crystallographic data of compound 3E; CCDC 2065484. SUPPLEMENTARY DATA 2 crystallographic data of compound 3L; CCDC 2065489. SOURCE DATA SOURCE DATA FIG.

2 Statistical source data of 40 previously reported variants. SOURCE DATA FIG. 4 Statistical source data of substrate scope. SOURCE DATA FIG. 5 Statistical source data of OD test. RIGHTS

AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Liu, Z., Calvó-Tusell, C., Zhou, A.Z. _et al._ Dual-function enzyme catalysis for enantioselective

carbon–nitrogen bond formation. _Nat. Chem._ 13, 1166–1172 (2021). https://doi.org/10.1038/s41557-021-00794-z Download citation * Received: 14 April 2021 * Accepted: 23 August 2021 *

Published: 18 October 2021 * Issue Date: December 2021 * DOI: https://doi.org/10.1038/s41557-021-00794-z SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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