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(BBSRC DTP) Functional characterisation of orphan cytochrome P450 enzymes in Mycobacterium tuberculosis


Project Description

Mycobacterium tuberculosis (Mtb) poses a major threat to human health worldwide and remains the biggest killer of humans among infectious diseases. It is typically treated using the drugs isoniazid and rifampicin, but many strains of Mtb have evolved such that they have become resistant to one or more drugs. These drug-resistant strains pose serious issues for infected patients, and the problem is compounded by the further development and spread of multidrug resistant (MDR), extremely drug resistant (XDR) and totally drug resistant (TDR) Mtb strains. It is clear that new strategies are required to address the global burden of drug-resistant Mtb. In recent studies, our group has identified cytochrome P450 (P450) enzymes as potential new drug targets in Mtb. We have expressed, characterised and crystallised several of the Mtb P450 enzymes. Among these are the P450s (CYPs) CYP142 and CYP125 that initiate the oxidative breakdown of the cholesterol side chain that facilitates the use of cholesterol as a carbon source for the bacterium1-3; and CYP121 that catalyses the formation of a C-C bond in the cyclic dipeptide cyclodityrosine, producing the secondary metabolite mycocyclosin. CYP121 is an essential gene in Mtb and a potentially important drug target4,5. While our group and others have cloned and expressed a number of the 20 P450s found in Mtb, the majority of these monooxygenase enzymes remain as “orphans” without known functions in the bacterial cell. The purpose of this DTP application is for a PhD appointee to express and purify the remaining “orphan” P450s, leading to the characterisation of Mtb P450 enzyme functions and to the structural analysis of these P450s in their ligand-free and substrate-bound forms. Through the detailed characterisation of these orphan P450s, the functions of these enzyme should become clear, enabling deeper insights into their roles in the cell. Other key work to be done will be the expression and purification of redox partner systems (specifically ferredoxin and ferredoxin proteins), leading to studies that demonstrate efficient catalytic interactions between the P450 enzymes and their redox partner systems. Many of the orphan Mtb P450 enzymes clearly have important roles in the Mtb cell, and this project should reveal novel functions for these enzymes, and in so doing should open new avenues that will lead to new discoveries and novel insights into functions of the orphan P450s.

http://www.mib.ac.uk
https://www.research.manchester.ac.uk/portal/Sam.Hay.html
https://www.research.manchester.ac.uk/portal/david.leys.html
https://www.research.manchester.ac.uk/portal/andrew.munro.html

Entry Requirements:
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Partnership. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP website View Website

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

1. McLean KJ et al. (2010), The Mycobacterium tuberculosis cytochromes P450: physiology, biochemistry & molecular intervention. Future Med. Chem. 2, 1339-53.
2. Driscoll MD et al. (2010), Structural and biochemical characterization of Mycobacterium tuberculosis CYP142: evidence for multiple cholesterol 27-hydroxylase activities in a human pathogen. J. Biol. Chem. 285, 38270-82.
3. McLean KJ et al. (2009), The structure of Mycobacterium tuberculosis CYP125: molecular basis for cholesterol binding in a P450 needed for host infection. J. Biol. Chem. 284, 35524-33.
4. El-Wahab et al. (2018), Design, synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics. Bioorg. Med. Chem. 26, 161-76.
5. Leys et al. (2003) Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 Å reveals novel features of cytochrome P450. J. Biol. Chem. 278, 5141-7.

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