Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9

Abstract

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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 P450BM3 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 P450BM3 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.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…This BM3 mutant was also shown to produce 2-hydroxyibuprofen (7a) from ibuprofen (7) (Tsotsou et al, 2012); however, experiments were only done in vitro or in a microtiter plate. Likewise, the production of product 7a in a preparative in vitro scale was described yielding 74.3 mg of product 7a (96% conversion) (Rentmeister et al, 2011). However, in this study, we were able to produce product 7a in a more relevant, biotechnological way using an E. coli-based whole-cell bioconversion system for CYP267B1 consisting of autologous redox partners and necessary cofactors within the cells.…”

Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

“…The variants generated up to now cover a wide range of structurally different drugs and often give rise to different metabolites, summarized in Table 1, with different yield. The availability of several crystal structures of P450 BM3 mutants [ 47] gives also the opportunity to perform docking simulations and to have important information not only on how to further improve the biocatalyst but also, more generally, on the structure-function relationship of the enzyme.…”

“…Other variants able to convert ibuprofen were generated by directed evolution [ 47]. These variants showed also a new activity toward desmethylnaproxen that is the human metabolite of naproxen, another substrate of CYP2C9 in vivo [ 47].…”

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

“…[19][20][21] Engineered P450 BM3 mediated hydroxylation of NSAIDs like diclofenac, ibuprofen and naproxen have been reported. [22][23][24][25] In the present study, fenamic acid NSAIDs (Figure 1) have been used as examples to demonstrate the ability of P450 BM3 mutants to produce human relevant metabolites. The aim of this study is to identify P450 BM3 variants capable of selective benzylic and aromatic hydroxylation with minimal secondary oxidation of fenamic acid NSAIDs that can be used as biocatalysts for large-scale drug metabolite production.…”