Production of high value drug metabolites using engineered cytochromes P450

Abstract

Production of drug metabolites is an area of biotechnology where manufacture bycytochromes P450 (P450s) offers significant advantages over synthetic methods. P450 BM3from Bacillus megaterium is an ideal candidate for metabolite synthesis. It is a soluble fusionof a P450 to its partner (cytochrome P450 reductase) with the fastest reaction rate for anyP450 in oxidising fatty acids. BM3 is structurally very well understood, and has been studiedextensively by random mutagenesis, with the aim of generating high activity oxidase variantsfor biotechnological uses. Several mutations occur frequently in such variants, but there islittle understanding of how common mutations underpin major alterations in BM3 substrateselectivity and activity. This thesis provides new information for BM3 Ala82 and Phe87variants, two of the most commonly mutated residues, to explain their impact on definingnovel oxidase function using a combination of structural and solution data. It is shown thattwo common conformations in BM3 P450 structures - the substrate free (SF) and substratebound (SB) states - are in an equilibrium (regardless of binding of substrates), and thatthermodynamic stability of the enzyme is key to their energetic separation. The A82Fmutation destabilises SF, allowing easier access to SB, and generating a more malleableP450 that binds diverse compounds. The F87V mutation removes steric bulk from the activesite, allowing easier access for non-natural substrates to bind in a less strained conformationabove the P450 heme. In isolation and combination, these mutations induce major changesin BM3 catalytic properties. The gastric proton pump inhibitor (PPI) drug omeprazole bindstightly to BM3 A82F variants. X-ray crystal structures show that the 5-methyl group of thePPIs omeprazole/esomeprazole is close to the heme, providing the first BM3 structure in an"active" conformation for substrate binding position. These PPIs are oxidized on the 5-methylgroup. The main human PPI metabolising enzymes are CYP2C19 and CYP3A4. Our datashow that the CYP2C19 metabolites of omeprazole, esomeprazole and rabeprazole are alsothose produced by BM3 variants, and with lansoprazole the major metabolite is also that forCYP3A4. Expanding these studies to other human P450 marker drug substrates revealedthat, by differential scanning fluorimetry (DSF) and electron paramagnetic resonance (EPR),multiple binding interactions occur between variant BM3 P450s and drugs such asdiclofenac and dextromethorphan. Although drug substrates used are markers for diversehuman P450s, product analysis showed that BM3 variants produced mainly a human drugmetabolite in each case. This suggests specific oxidation reactions are favoured for P450s,and occur preferentially when substrates are mobile in P450 active sites. Our work showsthat certain "gatekeeper" mutations in BM3 create conformational flexibility by destabilisingthe SF versus the SB state. This provides novel routes to screen for altered activity by usingstability, rather than the more constrained single activity screening techniques.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)