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Dual-function enzyme catalysis for enantioselective carbon–nitrogen bond formation

Liu, Zhen and Calvó-Tusell, Carla and Zhou, Andrew Z. and Chen, Kai and Garcia-Borràs, Marc and Arnold, Frances H. (2021) Dual-function enzyme catalysis for enantioselective carbon–nitrogen bond formation. Nature Chemistry, 13 (12). pp. 1166-1172. ISSN 1755-4330. doi:10.1038/s41557-021-00794-z.

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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.

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Liu, Zhen0000-0002-1119-0693
Calvó-Tusell, Carla0000-0003-2681-8460
Zhou, Andrew Z.0000-0002-1763-7353
Chen, Kai0000-0002-3325-3536
Garcia-Borràs, Marc0000-0001-9458-1114
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© The Author(s), under exclusive licence to Springer Nature Limited 2021. Received 14 April 2021; Accepted 23 August 2021; Published 18 October 2021. 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. 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 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. Author 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. 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. 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.
Group:Resnick Sustainability Institute
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-19-2-0026
Ministerio de Ciencia e Innovación (MCINN)PID2019-111300GA-I00
Generalitat de Catalunya2018-BP-00204
Resnick Sustainability InstituteUNSPECIFIED
Barcelona Supercomputing Center (BSC)RES-QSB-2020-2-0016
Subject Keywords:Biocatalysis; Computational chemistry
Issue or Number:12
Record Number:CaltechAUTHORS:20210421-171106293
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Official Citation: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).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108797
Deposited By: Tony Diaz
Deposited On:23 Apr 2021 18:22
Last Modified:03 Dec 2021 17:18

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