CaltechAUTHORS
  A Caltech Library Service

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. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20210421-171106293

[img] PDF - Submitted Version
Creative Commons Attribution Non-commercial No Derivatives.

3MB
[img] PDF - Supplemental Material
Creative Commons Attribution Non-commercial No Derivatives.

29MB

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20210421-171106293

Abstract

Whereas enzymatic asymmetric carbene N–H insertion is a powerful method for preparation of chiral amines in principle, it has suffered from limited enantioselectivity in practice. In this work, we demonstrate that engineered cytochrome P450 enzymes can catalyze this abiological C–N bond-forming reaction with excellent activity and selectivity (up to 32,100 TTN, >99% yield and 98% e.e.) to prepare a series of bioactive α-amino lactones, which have not been accessed previously using a carbene insertion strategy. The enzymes are dual-function catalysts, effecting both carbene transfer and enantioselective proton-transfer catalysis, in a single active site. To gain insight into the mechanism of the enzymatic transformation, especially in the asymmetric protonation step, we performed extensive molecular dynamics simulations and density functional theory (DFT) calculations. Computational studies uncover the important roles of active-site residues that enable high activity and selectivity through interacting with the carbene intermediate and the amine substrate, and directing water molecules for selective proton transfer.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.26434/chemrxiv.14452158.v1DOIDiscussion Paper
ORCID:
AuthorORCID
Liu, Zhen0000-0002-6313-823X
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:Licence: CC BY-NC-ND 4.0. Preprint submitted on 20.04.2021, 06:07 and posted on 20.04.2021. This work was supported by the NSF 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 Pinos 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 David C. Miller, Sabine Brinkmann-Chen, Ravi Lal, and Tian Zeng (May) for helpful discussions and comments on the manuscript. We further thank Mona Shahgholi for HRMS analysis and Michael K. Takase for X-ray crystallographic analysis. 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: A provisional patent application based on this manuscript has been filed through the California Institute of Technology. Data availability: All data necessary to support the paper’s conclusions are available in the main text and the Supporting 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.
Group:Resnick Sustainability Institute
Funders:
Funding AgencyGrant Number
NSFMCB-2016137
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
Record Number:CaltechAUTHORS:20210421-171106293
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210421-171106293
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108797
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:23 Apr 2021 18:22
Last Modified:24 Jun 2021 23:46

Repository Staff Only: item control page