Synthetic biology applications of P450 BM3

The cytochromes P450 (P450s) have crucial human physiological functions; being responsible for several key oxidative transformations of sex steroids as well as being primary enzymes involved in xenobiotic detoxification. These reactions involve P450-dependent activation of O2 on a heme iron and insertion of an oxygen atom into the substrate. This usually occurs at a specific position on a given substrate (e.g. giving hydroxylation, though other outcomes are feasible), whereas chemical oxidation of such substrates often results in several different products. The human P450s are membrane-bound, often unstable and interact with membrane-associated redox partners. However, higher activity, soluble P450s are found in bacteria. The catalytically most efficient of these oxidases are P450s naturally fused to a NADPH-dependent reductase. The best studied is the biotechnologically important P450 BM3 with turnover numbers of ~300/s with fatty acids. We have engineered BM3 by mutagenesis of its P450 domain to facilitate conformational reorganization; and in so doing produced variants with dramatically altered substrate recognition. These include mutants that oxidize steroids at distinct, mutant-specific positions; and ones generating metabolites
of human drugs oxidized in the same positions as done by their major human P450 catalysts. The main objectives of the studentship are to exploit BM3 mutant enzymes to make oxidized metabolites of steroids (progesterone, testosterone) and selected drugs. Our preliminary published work shows the viability of this plan (e.g. for omeprazole), and in unpublished work we have seen metabolites from a number of substrates (e.g. dextromethorphan) using conformationally perturbed BM3 mutant catalysts. The student will generate/purify BM3 variants (including A82F mutants); thereafter characterizing interactions with a range of steroids and pharmaceuticals by optical (heme) titrations and steady-state kinetics. In collaboration with Agilent, products of oxidative metabolism with all substrates will be analyzed using state-of-the-art LC or GC-MS/MS facilities to identify oxidized products and quantify their formation and the extent to which NADPH oxidation is coupled to product formation. Crystal structures of heme domains of relevant BM3 mutants that produce drug/steroid metabolites of interest (e.g. human metabolites) will be determined, and
complexes with the substrates sought by co-crystallization and/or soaking of ligand-free crystals. Using structural data and/or molecular modelling embracing predicted binding mode (based on position of drug/steroid oxidation), BM3 rational engineering will be done to improve binding affinity and/or regioselectivity of oxidation as required. At Agilent, product profiles of 2nd generation variants will be interrogated and scale-up studies for key metabolite preparation done, using products from in vitro turnover studies with pure mutant enzymes, and comparing efficiency with that from E. coli transformant extracts or whole cells. The student will be trained in protein engineering, enzyme isolation/characterization, spectroscopy and structural biology; as well as in organic product analysis, quantification and preparation. The project exploits complementary expertise at the academic and industrial sites, and the student will engage in an interdisciplinary project aimed at important synthetic biology applications of BM3 in making
valuable human steroid/drug metabolites.