Abstract
Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis (Mtb), the causative agent, that are resistant to first- and second-line antitubercular drugs urges the development of new therapeutics. The genome of Mtb encodes 20 cytochrome P450 enzymes, at least some of which are potential candidates (CYP121, CYP125, and CYP128) for drug targeting. In this regard, we examined the specific role of CYP125 in the cholesterol degradation pathway, using genetic and mass spectrometric approaches. The analysis of lipid profiles from Mtb cells grown on cholesterol revealed that CYP125, by virtue of its C26-monooxygenase activity, is essential for cholesterol degradation, and, consequently, for the incorporation of side-chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl-branched phthiocerol dimycocerosates (PDIM). Moreover, this work also led to the identification of cholest-4-en-3-one as a source of cellular toxicity. Herein, we describe the experimental procedures that led to elucidation of the physiological function of CYP125. A similar approach can be used to study other important Mtb P450 enzymes.
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Acknowledgment
This work was supported by NIH R01 grants AI074824 (to PROM) and AI51667 (to J.S.C.) and NIH NCRR grants RR01614 and RR019934 (to A.L.B).
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Ouellet, H., Chow, E.D., Guan, S., Cox, J.S., Burlingame, A.L., de Montellano, P.R.O. (2013). Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium tuberculosis . In: Phillips, I., Shephard, E., Ortiz de Montellano, P. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 987. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-321-3_7
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DOI: https://doi.org/10.1007/978-1-62703-321-3_7
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-321-3
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