Bacterial P450 engineering for production of valuable drug metabolites

The US Food and Drug Administration (FDA) have recommended that many human drug metabolites (as well as their parent drugs) should be subject to testing for safety/toxicity. The primary enzymes in human drug metabolism are the cytochromes P450, which catalyze oxidative metabolism of numerous human drugs and other xenobiotics. However, since human P450s and their reductases are relatively slow and unstable membrane-bound enzymes, they are unlikely to be useful in pharmaceutical metabolite production. Similarly, chemical synthesis of complex drug metabolites is laborious and the chemical oxidation of drugs is likely to be non-specific and to produce various side-products in addition to any of the desired "human-type" metabolite. This PhD project will address this problem by use of a high-activity, soluble bacterial P450 enzyme (P450 BM3 from Bacillus megaterium - an efficient P450-P450 reductase fusion enzyme). BM3 is an intensively studied enzyme due to its catalytic proficiency, and has the highest catalytic activity of any P450 enzyme (285 s-1 for oxidation of arachidonic acid). Our recent work has produced BM3 "gatekeeper" variants in which BM3's substrate selectivity profile is dramatically altered from that of the wild-type enzyme, due to mutations that introduce greater P450 conformational flexibility and a larger active site volume close to the heme. In underpinning work, we have shown that these gatekeeper mutants can bind/metabolise numerous substrates that are not recognized by wild-type BM3. These include pharmaceuticals such as the anti-diabetic troglitazone and the gastric proton pump inhibitor omeprazole, as well as steroids (e.g. testosterone). Importantly, many of the BM3 metabolites made are the same as those from the main human drug metabolising P450s. This shows that the highly efficient BM3 gatekeeper mutants could provide an important route to solving the issue of how to produce human drug metabolites in useful amounts for drug safety testing. This project will offer the student extensive training at the MIB in Manchester (focusing on molecular biology; protein expression/engineering; compound screening and identification of new drug substrates; enzyme turnover and analytical studies (using NMR, HPLC- and GC-MS) to characterize oxidized products formed from novel substrates identified by high-throughput compound screening; and BM3 gatekeeper mutant crystallization in novel substrate-bound forms using X-ray diffraction methods). In preliminary work we have already identified new substrates for the BM3 gatekeeper mutants by titrating the potential substrates against BM3 gatekeeper mutants and identifying the formation of a high-spin P450 heme iron species - a typical assay used to identify new P450 substrates and to quantify (by UV-vis spectroscopic titration) their affinity (Kd value). Studies at Cypex Ltd will focus on fermentation of BM3 mutant expression cells permeabilised to facilitate drug entry, and analytical studies to identify and quantify key drug metabolites formed. The project will provide the student with broad training in enzymology, structural biology, microbiology and analytical techniques, and will also equip the student with important skills relevant for a career in industry or academia. The project aligns with BBSRC remit in priority areas such as "Technology Development for the Biosciences" (through development of high-throughput screens for substrate identification and characterization) and "Industrial Biotechnology and Bioenergy" (through work to identify and characterize human drug metabolites produced in good yield by BM3 gatekeeper mutants). The project also aligns with the DTP theme of "World Class Underpinning Biosciences" (via key research to develop efficient routes to production of bona fide human drug metabolites using BM3 mutants, with the ultimate aim of producing large amounts of these metabolites for applications including drug safety testing).