The World Health Organization regards tuberculosis as a world pandemic disease. There is increased demand for new drugs to tackle this threat. This threat has been further elevated with the emergence of drug resistant strains of the causative pathogen, Mycobacterium tuberculosis (Mtb), thereby increasing the urgency for development of novel anti-tubercular drugs. Success in whole genome sequence determination of Mtb revealed a large cohort of cytochrome P450 (CYP) enzymes. Research on these Mtb P450s has shown that several of them are critical to the survival of the pathogen. CYP121A1 and CYP128A1 have been demonstrated to be essential using knockout experiments. CYP125A1 and CYP142A1 have been shown to play crucial roles in bacterial catabolism of host steroids, with CYP125A1 also shown to be located within a gene cluster highly important for bacterial virulence and infectivity. CYP144A1 was shown to be one of the genes whose expression is elevated when Mtb was exposed to macrophage-like conditions, and gene knockout studies using the H37Rv virulent strain of Mtb indicated the ΔCYP144A1 mutant to be more sensitive to the clotrimazole antifungal. CYP126A1 was shown to be located within a cluster of genes highly important for the de novo synthesis of purines in Mtb. These and other data suggested these enzymes to be important to the growth process of Mtb and thus potential drug targets for developing novel therapeutics. Findings in this PhD have revealed that many characteristics of CYP144A1 and CYP126A1 are comparable to previous Mtb P450s reported to date. CYP144A1 is highly conserved within the Mycobacterium genus and specifically within pathogenic species. Transcriptomic analysis has revealed an alternative truncated transcript leading to the production of two physiologically relevant versions of CYP144A1. Our comparative biophysical characterization of both versions (CYP144A1-FLV and -TRV) show both enzymes to be similar in their binding tightly to azole antifungals. EPR and DSC studies show that the 30 amino acid truncation (to form CYP144A1-TRV) does not affect the heme electronic environment and the overall thermal stability of the enzymes. X-ray crystallography was utilized to determine the first crystal structure of a Mtb CYP144 family enzyme. The structure reveals that CYP144A1 possesses a large hydrophobic active site primed for accommodating large hydrophobic substrates. Further chemoproteomic profiling identified novel compounds, which bind in both inhibitor-like and substrate-like modes to CYP144A1, resulting in the development of novel CYP144A1 compounds for use as chemical probes for this P450. Fragment and compound screening identified several ligands with varying binding affinities for CYP126A1, suggesting that this P450 is capable of binding and catalyzing reactions with a wide range of substrates. Turnover experiments proved catalytic activity of CYP126A1 on one of these compounds (Compound 4). Crystallization of CYP126A1 with various compound “hits” (compounds 1 and 7, the azole drug ketoconazole) revealed involvement of several important residues within the active site of CYP126A1 in interactions with these molecules, thus providing important information for designing inhibitors for this enzyme. Both CYP144A1 and CYP126A1 display important characteristics that contribute to our general understanding of cytochromes P450 as a whole, and of Mtb P450s in particular. This PhD project has established the first instance of leaderless transcripts in Mtb P450s and has presented the first crystal structures of both CYP144A1 and CYP126A1, as well as identifying novel, useful chemicals that can be used as mechanistic probes for these enzymes as well as providing the basis for Mtb P450 isoform-specific inhibitors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:694267 |
| Date | January 2016 |
| Creators | Chenge, Jude |
| Contributors | Munro, Andrew ; Leys, David |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigating-orphan-cytochrome-p450s-in-mycobacterium-tuberculosis-insights-into-enzyme-structure-function-and-inhibitor-design(2965ac43-d907-41d8-8222-1f327dc745e4).html |
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