Flavocytochrome P450 BM3 engineering for scalable P450 reactions

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Generation of oxyfunctionalized organic chemicals with regio/stereoselective introduction of oxygen atoms is highly desirable for industrial manufacture of high value chemicals, e.g. costly intermediates, drug metabolism standards, bioactive lipids, steroids etc. However, traditional methods are chemically challenging with significant problems associated with lack of positional selectivity, use of harsh reagents and processing. Enzyme chemistry offers alternatives to such synthetic approaches, and cytochrome P450 (P450) monooxygenases, in particular, can introduce oxygen atoms into substrates with a high degree of regio- and stereoselectivity. The major aim of this CASE studentship proposal is to engineer a high activity P450-P450 reductase fusion enzyme (flavocytochrome P450 BM3; BM3) to perform specific oxygenations leading to high value synthetic intermediates and oxygenated products for biotechnological exploitation, as described below: BM3 is an ideal P450 system for biotechnological applications. It is a soluble, single component enzyme with activity ~1000-fold that of multicomponent, membrane-bound eukaryotic P450 systems. We have a firm understanding of BM3's structure and residues controlling substrate selectivity/catalysis. Preceding studies from Munro and other groups have proven its 'malleability' and the ability to re-engineer its substrate selectivity and catalytic properties by both rational mutagenesis and semi-random directed evolution with screening for desired activities. This project aims to isolate BM3 variants that catalyse enantio- and regioselective hydroxylations of active pharmaceutical ingredients and/or their intermediates. Hydroxylations will be focused at positions of medium activation, such as benzylic, allylic and tertiary carbon centres. Important targets include terminal alkenes, and the development of selective BM3 mutants for either epoxidation or allylic hydroxylation. In these cases, we will use enzymes with or without F87G mutations, where the Phe87 phenyl side chain protects the terminal C=C double bond and favours hydroxylation at the adjacent carbon. The following approaches will be used: At Manchester, the student will investigate substrate-binding and turnover using an existing panel of BM3 point mutants, and employing pre-existing libraries of mutants (typically 1-2 residues changed per mutant) in different P450 regions known as substrate recognition sequences (SRS) that line the active site and contain substrate interacting residues. Libraries will be screened using BBSRC-sponsored robotic facilities at Manchester, and using expression cell extracts and optical/fluorescence screens based on (i) substrate-dependent NADPH oxidation and (ii) substrate-linked oxygen consumption by 'OxoPlate' technology, that we have already validated using model substrates. Mutants with desired activities will be validated using substrate binding (by heme absorbance, ITC) and turnover assays. Beneficial mutations will be combined (e.g. with ones from other SRS regions) and secondary screens used as appropriate to further optimize substrate recognition/turnover. Heme (P450) domains of relevant mutants will be purified/crystallized, enabling their structural elucidation in substrate-bound forms to rationalize binding mode and to allow further 'polishing' by rational mutagenesis to improve the desired reactions. Optimized mutants will be analyzed for efficiency of product formation at Pfizer, using both in vitro (pure enzyme) and cell culture systems, leading to studies of product profiles, system optimization and scale-up to generate quantities of desired oxy-products in amounts suited to industrial applications. The student will receive a broad training in protein engineering, enzyme biochemical, biophysical & structural analysis; combined with organic chemical characterization and industrial scale-up technology to facilitate biotechnological applications for the targeted oxygenated products.