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.

We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a "surrogate" for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping.

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