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Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9
Author(s) -
Rentmeister Andrea,
Brown Tristan R.,
Snow Christopher D.,
Carbone Marti.,
Arnold Frances H.
Publication year - 2011
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201000452
Subject(s) - bacillus megaterium , chemistry , enzyme , active site , metabolite , biochemistry , naproxen , drug metabolism , cytochrome p450 , combinatorial chemistry , stereochemistry , biology , bacteria , medicine , genetics , alternative medicine , pathology
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 CH bonds, a transformation that plays a key role in metabolism; bioconversions using P450 enzymes have emerged as a powerful alternative. Variants of P450 BM3 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.