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Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes

Renata, Hans and Lewis, Russell D. and Sweredoski, Michael J. and Moradian, Annie and Hess, Sonja and Wang, Z. Jane and Arnold, Frances H. (2016) Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes. Journal of the American Chemical Society, 138 (38). pp. 12527-12533. ISSN 0002-7863. PMCID PMC5042878. doi:10.1021/jacs.6b06823. https://resolver.caltech.edu/CaltechAUTHORS:20160919-123244987

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Abstract

Following the recent discovery that heme proteins can catalyze the cyclopropanation of styrenyl olefins with high efficiency and selectivity, interest in developing new enzymes for a variety of non-natural carbene transfer reactions has burgeoned. The fact that diazo compounds and other carbene precursors are known mechanism-based inhibitors of P450s, however, led us to investigate if they also interfere with this new enzyme function. We present evidence for two inactivation pathways that are operative during cytochrome P450-catalyzed cyclopropanation. Using a combination of UV–vis, mass spectrometry, and proteomic analyses, we show that the heme cofactor and several nucleophilic side chains undergo covalent modification by ethyl diazoacetate (EDA). Substitution of two of the affected residues with less-nucleophilic amino acids led to a more than twofold improvement in cyclopropanation performance (total TTN). Elucidating the inactivation pathways of heme protein-based carbene transfer catalysts should aid in the optimization of this new biocatalytic function.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/jacs.6b06823DOIArticle
http://pubs.acs.org/doi/suppl/10.1021/jacs.6b06823PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042878PubMed CentralArticle
ORCID:
AuthorORCID
Renata, Hans0000-0003-2468-2328
Lewis, Russell D.0000-0002-5776-7347
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Hess, Sonja0000-0002-5904-9816
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2016 American Chemical Society. Received: July 1, 2016; Publication Date (Web): August 30, 2016. The authors thank Dr. Andrew R. Buller, Dr. Christopher K. Prier, Dr. David K. Romney, and Dr. Sabine Brinkmann-Chen for helpful comments on the manuscript. We thank the staff of the Proteome Exploration Laboratory at Caltech, especially Roxana Eggleston-Rangel, for assistance with the proteomic analyses, Dr. Scott Virgil and the Caltech Center for Catalysis and Chemical Synthesis (3CS) for assistance with SFC and HRMS analyses, and the Beckman Institute Laser Resource Center (BILRC) at Caltech for use of their CD spectrometer. This work was supported by the Gordon and Betty Moore Foundation through grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative, and the National Science Foundation, Office of Chemical, Bioengineering, Environmental and Transport Systems SusChEM Initiative (Grant CBET-1403077). The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through Grant GBMF775, and the Beckman Institute. R.D.L. is supported by NIH/NRSA training grant (5 T32 GM07616). The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF2809
NSFCBET-1403077
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
NIH Predoctoral Fellowship5 T32 GM07616
Issue or Number:38
PubMed Central ID:PMC5042878
DOI:10.1021/jacs.6b06823
Record Number:CaltechAUTHORS:20160919-123244987
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160919-123244987
Official Citation:Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes Hans Renata, Russell D. Lewis, Michael J. Sweredoski, Annie Moradian, Sonja Hess, Z. Jane Wang, and Frances H. Arnold Journal of the American Chemical Society 2016 138 (38), 12527-12533 DOI: 10.1021/jacs.6b06823
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:70429
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:28 Sep 2016 18:37
Last Modified:19 Apr 2022 17:02

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