Skip to main content

Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium tuberculosis

Part of the Methods in Molecular Biology book series (MIMB,volume 987)

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.

Key words

  • Mycobacterium tuberculosis
  • Cytochrome P450
  • Specialized transduction
  • Mycobacteriophage
  • Total lipid
  • Cholesterol
  • LTQFT/MS
  • LC–MS

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-62703-321-3_7
  • Chapter length: 16 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   119.00
Price excludes VAT (USA)
  • ISBN: 978-1-62703-321-3
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   159.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Cole ST et al (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544

    PubMed  CrossRef  CAS  Google Scholar 

  2. Bhatt A, Fujiwara N, Bhatt K, Gurcha SS, Kremer L, Chen B, Chan J, Porcelli SA, Kobayashi K, Besra GS, Jacobs WR Jr (2007) Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proc Natl Acad Sci U S A 104:5157–5162

    PubMed  CrossRef  CAS  Google Scholar 

  3. Bhatt A et al (2007) The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development. Mol Microbiol 64:1442–1454

    PubMed  CrossRef  CAS  Google Scholar 

  4. Jackson M, Stadthagen G, Gicquel B (2007) Long-chain multiple methyl-branched fatty acid-containing lipids of Mycobacterium tuberculosis: biosynthesis, transport, regulation and biological activities. Tuberculosis (Edinb) 87:78–86

    CrossRef  CAS  Google Scholar 

  5. Mahadevan U, Padmanaban G (1998) Cloning and expression of an acyl-CoA dehydrogenase from Mycobacterium tuberculosis. Biochem Biophys Res Commun 244:893–897

    PubMed  CrossRef  CAS  Google Scholar 

  6. Ouellet H, Johnston JB, Montellano PR (2011) Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis. Trends Microbiol 19:530–539

    PubMed  CrossRef  CAS  Google Scholar 

  7. Van der Geize R et al (2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci U S A 104:1947–1952

    PubMed  CrossRef  Google Scholar 

  8. Minnikin DE, Kremer L, Dover LG, Besra GS (2002) The methyl-branched fortifications of Mycobacterium tuberculosis. Chem Biol 9:545–553

    PubMed  CrossRef  CAS  Google Scholar 

  9. Onwueme KC, Vos CJ, Zurita J, Ferreras JA, Quadri LE (2005) The dimycocerosate ester polyketide virulence factors of mycobacteria. Prog Lipid Res 44:259–302

    PubMed  CrossRef  CAS  Google Scholar 

  10. Jain M et al (2007) Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling. Proc Natl Acad Sci U S A 104:5133–5138

    PubMed  CrossRef  CAS  Google Scholar 

  11. Savvi S et al (2008) Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids. J Bacteriol 190:3886–3895

    PubMed  CrossRef  CAS  Google Scholar 

  12. Pandey AK, Sassetti CM (2008) Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci U S A 105:4376–4380

    PubMed  CrossRef  CAS  Google Scholar 

  13. Ouellet H et al (2010) Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one. Mol Microbiol 77:730–742

    PubMed  CrossRef  CAS  Google Scholar 

  14. Yang X et al (2009) Cholesterol metabolism increases the metabolic pool of propionate in Mycobacterium tuberculosis. Biochemistry 48:3819–3821

    PubMed  CrossRef  CAS  Google Scholar 

  15. Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100:12989–12994

    PubMed  CrossRef  CAS  Google Scholar 

  16. Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84

    PubMed  CrossRef  CAS  Google Scholar 

  17. Holsclaw CM et al (2008) Structural characterization of a novel sulfated menaquinone produced by stf3 from Mycobacterium tuberculosis. ACS Chem Biol 3:619–624

    PubMed  CrossRef  CAS  Google Scholar 

  18. de Carvalho LP et al (2010) Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates. Chem Biol 17:1122–1131

    PubMed  CrossRef  Google Scholar 

  19. de Carvalho LP et al (2010) Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. Chem Biol 17:323–332

    PubMed  CrossRef  Google Scholar 

  20. Bardarov S et al (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007–3017

    PubMed  CAS  Google Scholar 

  21. Shen CF, Hawari J, Kamen A (2004) Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents. J Chromatogr B Analyt Technol Biomed Life Sci 810:119–127

    PubMed  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

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

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-321-3_7

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-320-6

  • Online ISBN: 978-1-62703-321-3

  • eBook Packages: Springer Protocols