1887

Abstract

The genome sequence of has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14α-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of and of strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against (MIC values of <02 and 03 μM, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 12 and 365 μM, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of , which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in H37Rv P450s encoded by genes (the sterol demethylase CYP51) and (CYP121). However, the azoles had a higher affinity for CYP121, with values broadly in line with the MIC values for . This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an Δ strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in and , then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of that have already developed resistance to isoniazid and other front-line drugs.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-10-2937
2002-10-01
2020-09-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/10/1482937a.html?itemId=/content/journal/micro/10.1099/00221287-148-10-2937&mimeType=html&fmt=ahah

References

  1. Aoyama Y, Kudo M, Asai K, Okonogi K, Horiuchi T, Gotoh O., Yoshida Y. 2000; Emergence of fluconazole-resistant sterol 14-demethylase P450 (CYP51) in Candida albicans is a model demonstrating the diversification mechanism of P450. Arch Biochem Biophys379:170–171
    [Google Scholar]
  2. Bellamine A, Mangla A. T, Nes W. D., Waterman M. R. 1999; Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis. Proc Natl Acad Sci USA96:8937–8942
    [Google Scholar]
  3. Bentley S. D, Chater K. F, Cerdeno-Tarraga A.-M.. 40 other authors 2002; Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature417:141–147
    [Google Scholar]
  4. Betlach M. C, Kealey J. T, Ashley G. W., McDaniel R. 1998; Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin. Biochemistry37:14937–14942
    [Google Scholar]
  5. Bhatt A, Stewart G. R., Kieser T. 2002; Transposition of Tn 4560 of Streptomyces fradiae in Mycobacterium smegmatis. FEMS Microbiol Lett206:241–246
    [Google Scholar]
  6. Blanchard J. S. 1996; Molecular mechanisms of drug resistance in Mycobacterium tuberculosis. Annu Rev Biochem65:215–239
    [Google Scholar]
  7. Blattner F. R, Plunkett G. 3rd, Bloch C. A.. 14 other authors 1997; The complete genome sequence of Escherichia coli K-12. Science277:1453–1474
    [Google Scholar]
  8. Brennan P. J., Nikaido H. 1995; The envelope of the mycobacteria. Annu Rev Biochem64:29–63
    [Google Scholar]
  9. Cole S. T, Brosch R, Parkhill J.. 39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature393:537–544
    [Google Scholar]
  10. Cole S. T, Eiglmeier K, Parkhill J.. 41 other authors 2001; Massive gene decay in the leprosy bacillus. Nature409:1007–1011
    [Google Scholar]
  11. Cooksey R. C, Crawford J. T, Jacobs W. R. Jr, Shinnick T. M. 1993; A rapid method for screening antimicrobial agents for activities against a strain of Mycobacterium tuberculosis expressing firefly luciferase. Antimicrob Agents Chemother37:1348–1352
    [Google Scholar]
  12. Cupp-Vickery J. R, Han O, Hutchinson C. R., Poulos T. L. 1996; Substrate-assisted catalysis in cytochrome P450eryF. Nat Struct Biol3:632–637
    [Google Scholar]
  13. Cupp-Vickery J. R, Garcia C, Hofacre A., McGee-Estrada K. 2001; Ketoconazole-induced conformational changes in the active site of cytochrome P450eryF. J Mol Biol311:101–110
    [Google Scholar]
  14. Daffe M., Draper P. 1998; The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol39:131–203
    [Google Scholar]
  15. Fowler K. 2002; Transposon Mutagenesis of Streptomyces coelicolor A3(2) PhD thesis University of East Anglia, UK;
    [Google Scholar]
  16. Gibson T. J. 1984; Studies in the Epstein-Barr Virus Genome PhD thesis University of Cambridge; UK:
    [Google Scholar]
  17. Guardiola-Diaz H. M, Foster L.-A, Mushrush D., Vaz A. D. N. 2001; Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis : implications for the treatment of tuberculosis. Biochem Pharmacol61:1463–1470
    [Google Scholar]
  18. Gust B, Kieser T., Chater K. F. 2002; Redirect© Technology: PCR-Targeting System in Streptomyces coelicolor Norwich, UK: John Innes Centre;
    [Google Scholar]
  19. Heym B, Philipp W., Cole S. T. 1996; Mechanisms of drug resistance in Mycobacterium tuberculosis. Curr Top Microbiol Immunol215:49–69
    [Google Scholar]
  20. Hobbs G, Frazer C. M, Gardner D. C. J, Cullum J. A., Oliver S. G. 1989; Dispersed growth of Streptomyces in liquid culture. Appl Microbiol Biotechnol31:272–277
    [Google Scholar]
  21. Hoffman H. L., Rathbun R. C. 2002; Review of the safety and efficacy of voriconazole. Expert Opin Investig Drugs11:409–429
    [Google Scholar]
  22. Kieser T, Bibb M. J, Buttner M. J, Chater K. F., Hopwood D. A. 2002; Practical Streptomyces Genetics Norwich, UK: John Innes Foundation;
    [Google Scholar]
  23. Kunst F, Ogasawara N, Moszer I.. 48 other authors 1997; The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature390:249–256
    [Google Scholar]
  24. Lamb D. C, Kelly D. E, Manning N. J., Kelly S. L. 1998; A sterol biosynthetic pathway in Mycobacterium. FEBS Lett437:142–144
    [Google Scholar]
  25. Lander E. S, Linton L. M, Birren B.. 253 other authors 2001; Initial sequencing and analysis of the human genome. Nature409:860–921
    [Google Scholar]
  26. Lepesheva G. I, Podust L. M, Bellamine A., Waterman M. R. 2001; Folding requirements are different between sterol 14alpha-demethylase (CYP51) from Mycobacterium tuberculosis and human or fungal orthologs. J Biol Chem276:28413–28420
    [Google Scholar]
  27. McLean K. J, Cheesman M. R, Rivers S. L.. 9 other authors 2002; Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis . J Inorg Biochem in press
    [Google Scholar]
  28. Nelson D. R, Koymans L, Kamataki T.. 9 other authors 1996; P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics6:1–42
    [Google Scholar]
  29. Noble M. A, Miles C. S, Chapman S. K, Lysek D. A, Mackay A. C, Reid G. A, Hanzlik R. P., Munro A. W. 1999; Roles of key active-site residues in flavocytochrome P450 BM3. Biochem J339:371–379
    [Google Scholar]
  30. Peterson J. A, Lu J. Y, Geisselsoder J, Graham-Lorence S, Carmona C, Witney F., Lorence M. C. 1992; Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J Biol Chem267:14193–14203
    [Google Scholar]
  31. Phetsuksiri B, Baulard A. R, Cooper A. M, Minnikin D. E, Douglas J. D, Besra G. S., Brennan P. J. 1999; Antimycobacterial activities of isoxyl and new derivatives through the inhibition of mycolic acid synthesis. Antimicrob Agents Chemother43:1042–1051
    [Google Scholar]
  32. Poulos T. L, Finzel B. C., Howard A. J. 1987; High-resolution crystal structure of cytochrome P450cam. J Mol Biol195:687–700
    [Google Scholar]
  33. Saint-Joanis B, Souchon H, Wilming M, Johnsson K, Alzari P. M., Cole S. T. 1999; Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis. Biochem J338:753–760
    [Google Scholar]
  34. Sambrook J, Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Schuster I. 1993; Ergosterol synthesis: the major target on antimycotic therapy. In Medicinal Implications in Cytochrome P450 Catalysed Biotransformations pp147–185 Edited by Ruckpaul K., Rein H.. Berlin: Akademie Verlag;
    [Google Scholar]
  36. Snapper S. B, Melton R. E, Mustafa S, Kieser T., Jacobs W. R. Jr. 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol4:1911–1919
    [Google Scholar]
  37. Souter A, McLean K. J, Smith W. E., Munro A. W. 2000; The genome sequence of Mycobacterium tuberculosis reveals cytochromes P450 as novel drug targets. J Chem Technol Biotechnol75:933–941
    [Google Scholar]
  38. Studier F. W., Moffat B. A. 1986; Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol189:113–130
    [Google Scholar]
  39. Wengenack N. L., Rusnak F. 2001; Evidence for isoniazid-dependent free radical generation catalysed by Mycobacterium tuberculosis KatG, and the isoniazid-resistant mutant KatG(S315T). Biochemistry40:8990–8996
    [Google Scholar]
  40. Yoshida Y, Noshiro M, Aoyama Y, Kawamoto T, Horiuchi T., Gotoh O. 1997; Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: II. Evolutionary analysis of protein and gene structures. J Biochem122:1122–1128
    [Google Scholar]
  41. Young D. B., Cole S. T. 1993; Leprosy, tuberculosis, and the new genetics. J Bacteriol175:1–6
    [Google Scholar]
  42. Zhang W. J, Ramamoorthy Y, Kilicarslan T, Nolte H, Tyndale R. F., Sellers E. M. 2002; Inhibition of cytochromes P450 by antifungal imidazole derivatives. Drug Metab Dispos30:314–318
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-10-2937
Loading
/content/journal/micro/10.1099/00221287-148-10-2937
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error