"Humanizing" a catalytically efficient bacterial oxygenase for high high value metabolite production

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Therapeutic drugs are extensively metabolised in the body, with the main enzymes involved being cytochromes P450 (P450s). P450s recognize foreign molecules and catalyse oxygen insertion reactions to make them more polar/soluble, and to target them for further enzymatic modification and excretion. There is increasing recognition that metabolites formed by P450-mediated oxidation of drugs may be pharmacologically active. The FDA and other regulatory agencies require testing of major metabolites as well as parent drugs. However, production of such P450 metabolites is challenging due to difficulties involved in inserting oxygen atoms in a regio-/stereo-selective manner by synthetic chemistry, and since human P450s are typically slow/unstable membrane-bound enzymes that require another membrane-bound reductase to function. An alternative route to making substantial amounts of valuable drug metabolites involves engineering a more efficient P450 to recognize/oxidize these drugs. Such an enzyme is the bacterial P450 BM3, a soluble P450 naturally fused to its reductase and possessing the highest P450 catalytic rate (~1000-fold faster than human P450s). BM3 mutants that oxidize drugs including caffeine and omeprazole have been generated. This project will involve further improvement of existing variants and engineering of novel BM3 mutants to expand the substrate range to analgesics, steroids and other important compounds. The project will be done in the Manchester Institute of Biotechnology and will involve research including protein engineering, structural biology of P450-drug complexes and analysis of drug metabolites formed (using HPLC- and GC-MS methods). The project is an industrial collaboration with Cypex Ltd, who have extensive expertise in the area.