Cytochrome P450 BM3 Engineering for the Production of Valuable Human Drug Metabolites

Abstract

Cytochromes P450 (P450s) are accountable for ~75% of human xenobiotic metabolism. P450 BM3, from Bacillus megaterium, is an enzyme which exhibits some of the highest catalytic rates of any P450, owing to its unique fusion structure. P450 BM3 has been subject to extensive mutagenesis studies to expand its catalytic repertoire. BM3 wild- type preferentially binds and hydroxylates fatty acids, such as arachidonic acid. A BM3 A82F/F87V double mutant (BM3 DM) was produced within Munro Lab to increase substrate scope. Previous studies have found that this mutant is able to bind a range of structurally diverse drugs from an FDA-approved library. This prompts a significant spin- shift in the Soret peak in ~60 % of compounds, indicative of substrate binding. The US Food and Drug Administration (FDA) states that any existing and novel xenobiotic must submit a full metabolic profile for safety testing. Membrane- bound P450s are unstable and inefficient and so do not produce sufficient yields of metabolites needed for characterisation and safety testing. This project aims to utilise BM3 DM as a tool for the production of major human metabolites. Metabolites formed from members of the Glitazone drug class have been characterised by LC-MS/MS and NMR. For two members of this antidiabetic drug class, metabolite structures are proposed. Computational methods were also developed for the design of P450 BM3 for the production of specific metabolites. Further work to exploit BM3 and its mutants could lead to large- scale production of human drug metabolites for use in the pharmaceutical industry and could have applications in the drug development pipeline, potentially leading to novel lead compounds.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)