Structure-function studies on α-methylacyl-CoA racemase

: (Alternative Format Thesis)

Abstract

Abstract
Tuberculosis globally accounts for the most deaths (~1.3 million) of any infectious disease with a particularly high disease burden on developing countries. Current treatments for tuberculosis use a cocktail of antibiotics taken over a long period (~6 months), but the efficacy of these treatments is hampered by Mycobacterium tuberculosis multi-drug resistance, and survival and persistence within the host. The enzyme α-methylacyl-CoA racemase (MCR) mediates M. tuberculosis survival and persistence. MCR is part of a wider family of α-methylacyl-CoA racemase enzymes (known as AMACR, P504S) which are involved in branched-chain fatty acid metabolism. They catalyse the conversion of R-2-methylacyl-CoAs into a near 1-to-1 mixture of the (2R)- and (2S)-epimers, enabling further metabolism by β-oxidation. They are also involved in the pharmacological activation of some non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen.
MCR is critical for M. tuberculosis as it grants catabolic access to host branched-chain lipids and cholesterol which allows the mycobacterium to reinforce its cell wall and enter a latent phase which reduces the efficacy of antibiotics. The exact role of MCR in M. tuberculosis is still being elaborated, but several experiments have shown MCR is functionally important, with its loss resulting in diminished growth and toxicity from the accumulation of 2R- fatty acids. Due to the importance of MCR to M. tuberculosis’s survival and persistence within the host, it has been explored as a model to understand the AMACR racemization mechanism and as a drug target.
This thesis reports the molecular details needed to understand the MCR racemization mechanism and establishes a foundation for the rational design of selective inhibitors which could offer targeted therapeutic strategies against M. tuberculosis. A new protocol for the production and purification of large quantities of MCR suitable for biochemical and structural biology applications is described. A new crystallisation condition was identified for wild-type MCR and its three active site mutants and their structures (space group C2) solved using X-ray crystallography. A 2H-exchange 1H NMR assay showed MCR was able to catalyse α-proton exchange, and a colorimetric assay was used to determine kinetic parameters for these mutants. These structural and kinetic results showed the diminution of activity in MCR mutants was linked to disruption of key hydrogen bonding and water-mediated interactions within the active site.
Branched- and straight-chain acyl-CoA esters and 2-arylthiopropanoyl-CoA inhibitors were soaked into new wild-type MCR crystals (space group I422) and inhibitory potency measured using the MCR colorimetric assay. These studies provide a detailed understanding of how ligands bind to MCR, and a new catalytic mechanism is proposed.
Through these studies, a detailed understanding of the AMACR mechanism is obtained along with an efficient platform for producing MCR and leveraging it for target driven drug discovery. Building on this study, selective and potent MCR inhibitors could be developed, or new inhibitors screened for due to the more efficient protein production, these inhibitors would then be rapidly tested making future MCR and AMACR drug discovery campaigns more efficient.

Date of Award	25 Mar 2026
Original language	English
Awarding Institution	University of Bath
Supervisor	Ravi Acharya (Supervisor) & Matthew Lloyd (Supervisor)