Bacterial P450 Engineering for Production of Valuable Drug Metabolites

Abstract

The heme mono-oxygenase Cytochromes P450 are a promiscuous enzyme superfamily, ubiquitous in nature, that have been utilised as key biotechnological tools in the biosynthetic production of drug metabolites. One enzyme of interest is the catalytically self-sufficient CYP102A1 (P450 BM3, BM3), a bacterial P450 where a heme domain is fused via a linker to a reductase domain, facilitating rapid electron transfer. BM3's malleable active site, ease of production and promiscuity make it the ideal tool for the production of high value compounds; extensive literature studies have already led to the identification of many metabolites. In this thesis, an in-depth study of BM3 and the promiscuous variant BM3 DM is presented. Studies on the BM3-mediated metabolism structurally diverse drug classes has led to the identification of a plethora of metabolites. BM3 DM was found to produce metabolites at high conversions relative to BM3 WT for key drugs from the steroid, glitazone, meglitinide, fibrate, terpene lactone and Vitamin A derivative classes. BM3 DM metabolic profiles indicated that several metabolites were produced per drug at varying selectivities, with multiple oxidative biotransformations observed. Utilising BM3 DM to diversify 2 drug libraries led to the identification of 9 metabolites for a library exhibiting poor pharmacokinetic properties, and the production of 17 pantothenamide metabolites from 6 parent compounds that were previously shown to possess anti-infective properties. 2 pantothenamide metabolites displayed antiplasmodial activity in the low micromolar range. BM3 DM therefore can be successfully utilised in drug library diversification, as the production of diverse metabolites has the potential to yield a multitude of analogues with potential improved functionality. Additionally, protein NMR studies of the isolated BM3 FMN domain's quinone states and interactions with the heme domain led to further information on the domain interactions and structure of the reductase domain at different stages of catalysis.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor), David Leys (Supervisor) & Jonathan Waltho (Supervisor)