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Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9

Rentmeister, Andrea and Brown, Tristan R. and Snow, Christopher D. and Carbone, Martina N. and Arnold, Frances H. (2011) Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9. ChemCatChem, 3 (6). pp. 1065-1071. ISSN 1867-3880. doi:10.1002/cctc.201000452.

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Simple and universal methods for the preparation of human drug metabolites are required to produce quantities sufficient for their characterization and toxicity testing. Synthetic chemistry lacks general catalysts for selective oxidation of unactivated C[BOND]H bonds, a transformation that plays a key role in metabolism; bioconversions using P450 enzymes have emerged as a powerful alternative. Variants of P450BM3 from Bacillus megaterium act on diverse substrates, including drugs. Acidic substrates, such as the compounds metabolized by CYP2C9, which is one of three main hepatic human P450s, are not accepted by P450_(BM3) variants engineered to date. Herein, we report bacterial mimics of CYP2C9, which are active on two widely administered drugs, naproxen and ibuprofen, that are CYP2C9 substrates in vivo. These P450_(BM3) variants can also act on desmethylnaproxen, the human metabolite of naproxen, and convert it to the 1,4-naphthoquinone derivative. We analyzed the crystal structure of the heme domain of an early intermediate in the directed-evolution experiment. The active site mutation, L75R, which initially conferred activity on charged substrates, dramatically increased structural flexibility in the B′-helix. This increased flexibility, which was accompanied by a dramatic decrease in enzyme stability, may contribute to the variant’s ability to accept a broader range of substrates.

Item Type:Article
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URLURL TypeDescription
Rentmeister, Andrea0000-0002-3107-4147
Snow, Christopher D.0000-0002-7690-3519
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Received: December 19, 2010. Article first published online: 21 Apr. 2011. We thank Dr. J. T. Kaiser and P. Nikolovski for their excellent support in high-throughput crystallization, data collection, and crystallographic questions. We acknowledge M. Shahgholi and Dr. D. VanderVelde for assistance with LC-MS, HRMS, and 2D-NMR spectroscopy, respectively. The authors acknowledge support from the Department of Energy BES (grant no. DE-FG02-06ER15762), the National Institutes of Health ARRA (grant no. R01 GM068664-05A1Z), and the Army Research Office (grant no. W911NF0810227). A.R. thanks the DFG for a postdoctoral research fellowship. We also acknowledge the Gordon and Betty Moore Foundation for the support of the Molecular Observatory at Caltech. Operations at SSRL are supported by the US DOE and NIH.
Funding AgencyGrant Number
Departement of Energy (DOE)DE-FG02-06ER15762
NIHR01 GM068664-05A1Z
Army Research Office (ARO)W911NF0810227
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
American Recovery and Reinvestment Act (ARRA)UNSPECIFIED
Subject Keywords:Biocatalysis; biomimetic synthesis; directed evolution; enzymes; metabolism
Issue or Number:6
Record Number:CaltechAUTHORS:20111202-122055370
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Official Citation:Rentmeister, A., Brown, T. R., Snow, C. D., Carbone, M. N. and Arnold, F. H. (2011), Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9. ChemCatChem, 3: 1065–1071. doi: 10.1002/cctc.201000452
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:28284
Deposited On:05 Dec 2011 16:29
Last Modified:09 Nov 2021 16:55

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