Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jun 1;364(Pt 2):555–562. doi: 10.1042/BJ20011380

Sterol 14alpha-demethylase activity in Streptomyces coelicolor A3(2) is associated with an unusual member of the CYP51 gene family.

David C Lamb 1, Kay Fowler 1, Tobias Kieser 1, Nigel Manning 1, Larissa M Podust 1, Michael R Waterman 1, Diane E Kelly 1, Steven L Kelly 1
PMCID: PMC1222601  PMID: 12023899

Abstract

The annotation of the genome sequence of Streptomyces coelicolor A3(2) revealed a cytochrome P450 (CYP) resembling various sterol 14alpha-demethylases (CYP51). The putative CYP open reading frame (SC7E4.20) was cloned with a tetrahistidine tag appended to the C-terminus and expressed in Escherichia coli. Protein purified to electrophoretic homogeneity was observed to bind the 14-methylated sterols lanosterol and 24-methylene-24,25-dihydrolanosterol (24-MDL). Reconstitution experiments with E. coli reductase partners confirmed activity in 14alpha-demethylation for 24-MDL, but not lanosterol. An S. coelicolor A3(2) mutant containing a transposon insertion in the CYP51 gene, which will abolish synthesis of the functional haemoprotein, was isolated as a viable strain, the first time a CYP51 has been identified as non-essential. The role of this CYP in bacteria is intriguing. No sterol product was detected in non-saponifiable cell extracts of the parent S. coelicolor A3(2) strain or of the mutant. S. coelicolor A3(2) CYP51 contains very few of the conserved CYP51 residues and, even though it can catalyse 14alpha-demethylation, it probably has another function in Streptomyces. We propose that it is a member of a new CYP51 subfamily.

Full Text

The Full Text of this article is available as a PDF (332.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akhtar M., Alexander K., Boar R. B., McGhie J. F., Barton D. H. Chemical and enzymic studies on the characterization of intermediates during the removal of the 14alpha-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives. Biochem J. 1978 Mar 1;169(3):449–463. doi: 10.1042/bj1690449b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aoyama Y., Yoshida Y. Interaction of lanosterol to cytochrome P-450 purified from yeast microscomes: evidence for contribution of cytochrome P-450 to lanosterol metabolism. Biochem Biophys Res Commun. 1978 May 15;82(1):33–38. doi: 10.1016/0006-291x(78)90572-7. [DOI] [PubMed] [Google Scholar]
  3. Aoyama Y., Yoshida Y., Sonoda Y., Sato Y. Deformylation of 32-oxo-24,25-dihydrolanosterol by the purified cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from yeast evidence confirming the intermediate step of lanosterol 14 alpha-demethylation. J Biol Chem. 1989 Nov 5;264(31):18502–18505. [PubMed] [Google Scholar]
  4. Aoyama Y., Yoshida Y., Sonoda Y., Sato Y. Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation. J Biol Chem. 1987 Jan 25;262(3):1239–1243. [PubMed] [Google Scholar]
  5. Bak S., Kahn R. A., Olsen C. E., Halkier B. A. Cloning and expression in Escherichia coli of the obtusifoliol 14 alpha-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14 alpha-demethylases (CYP51) from fungi and mammals. Plant J. 1997 Feb;11(2):191–201. doi: 10.1046/j.1365-313x.1997.11020191.x. [DOI] [PubMed] [Google Scholar]
  6. Bard M., Lees N. D., Turi T., Craft D., Cofrin L., Barbuch R., Koegel C., Loper J. C. Sterol synthesis and viability of erg11 (cytochrome P450 lanosterol demethylase) mutations in Saccharomyces cerevisiae and Candida albicans. Lipids. 1993 Nov;28(11):963–967. doi: 10.1007/BF02537115. [DOI] [PubMed] [Google Scholar]
  7. Bellamine A., Mangla A. T., Nes W. D., Waterman M. R. Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):8937–8942. doi: 10.1073/pnas.96.16.8937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bloch K. The biological synthesis of cholesterol. Science. 1965 Oct 1;150(3692):19–28. doi: 10.1126/science.150.3692.19. [DOI] [PubMed] [Google Scholar]
  9. Chung S. T. Tn4556, a 6.8-kilobase-pair transposable element of Streptomyces fradiae. J Bacteriol. 1987 Oct;169(10):4436–4441. doi: 10.1128/jb.169.10.4436-4441.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S., Barry C. E., 3rd Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998 Jun 11;393(6685):537–544. doi: 10.1038/31159. [DOI] [PubMed] [Google Scholar]
  11. Cole S. T., Eiglmeier K., Parkhill J., James K. D., Thomson N. R., Wheeler P. R., Honoré N., Garnier T., Churcher C., Harris D. Massive gene decay in the leprosy bacillus. Nature. 2001 Feb 22;409(6823):1007–1011. doi: 10.1038/35059006. [DOI] [PubMed] [Google Scholar]
  12. Embley T. M., Stackebrandt E. The molecular phylogeny and systematics of the actinomycetes. Annu Rev Microbiol. 1994;48:257–289. doi: 10.1146/annurev.mi.48.100194.001353. [DOI] [PubMed] [Google Scholar]
  13. Guengerich F. P. Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems. Biochemistry. 1983 Jun 7;22(12):2811–2820. doi: 10.1021/bi00281a007. [DOI] [PubMed] [Google Scholar]
  14. Guengerich F. P. Reactions and significance of cytochrome P-450 enzymes. J Biol Chem. 1991 Jun 5;266(16):10019–10022. [PubMed] [Google Scholar]
  15. Jackson C. J., Lamb D. C., Kelly D. E., Kelly S. L. Bactericidal and inhibitory effects of azole antifungal compounds on Mycobacterium smegmatis. FEMS Microbiol Lett. 2000 Nov 15;192(2):159–162. doi: 10.1111/j.1574-6968.2000.tb09375.x. [DOI] [PubMed] [Google Scholar]
  16. Jefcoate C. R. Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. Methods Enzymol. 1978;52:258–279. doi: 10.1016/s0076-6879(78)52029-6. [DOI] [PubMed] [Google Scholar]
  17. Jenkins C. M., Waterman M. R. Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. J Biol Chem. 1994 Nov 4;269(44):27401–27408. [PubMed] [Google Scholar]
  18. Kalb V. F., Woods C. W., Turi T. G., Dey C. R., Sutter T. R., Loper J. C. Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA. 1987 Dec;6(6):529–537. doi: 10.1089/dna.1987.6.529. [DOI] [PubMed] [Google Scholar]
  19. Kelly S. L., Lamb D. C., Cannieux M., Greetham D., Jackson C. J., Marczylo T., Ugochukwu C., Kelly D. E. An old activity in the cytochrome P450 superfamily (CYP51) and a new story of drugs and resistance. Biochem Soc Trans. 2001 May;29(Pt 2):122–128. doi: 10.1042/0300-5127:0290122. [DOI] [PubMed] [Google Scholar]
  20. Kelly S. L., Lamb D. C., Kelly D. E., Loeffler J., Einsele H. Resistance to fluconazole and amphotericin in Candida albicans from AIDS patients. Lancet. 1996 Nov 30;348(9040):1523–1524. doi: 10.1016/S0140-6736(05)65949-1. [DOI] [PubMed] [Google Scholar]
  21. Lamb D. C., Kelly D. E., Kelly S. L. Molecular diversity of sterol 14alpha-demethylase substrates in plants, fungi and humans. FEBS Lett. 1998 Mar 27;425(2):263–265. doi: 10.1016/s0014-5793(98)00247-6. [DOI] [PubMed] [Google Scholar]
  22. Lamb D. C., Kelly D. E., Manning N. J., Kelly S. L. A sterol biosynthetic pathway in Mycobacterium. FEBS Lett. 1998 Oct 16;437(1-2):142–144. doi: 10.1016/s0014-5793(98)01218-6. [DOI] [PubMed] [Google Scholar]
  23. Lamb D. C., Kelly D. E., Schunck W. H., Shyadehi A. Z., Akhtar M., Lowe D. J., Baldwin B. C., Kelly S. L. The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem. 1997 Feb 28;272(9):5682–5688. doi: 10.1074/jbc.272.9.5682. [DOI] [PubMed] [Google Scholar]
  24. Lamb David, Kelly Diane, Kelly Steven. Molecular aspects of azole antifungal action and resistance. Drug Resist Updat. 1999 Dec;2(6):390–402. doi: 10.1054/drup.1999.0112. [DOI] [PubMed] [Google Scholar]
  25. Nes W. D., Norton R. A., Crumley F. G., Madigan S. J., Katz E. R. Sterol phylogenesis and algal evolution. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7565–7569. doi: 10.1073/pnas.87.19.7565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nes W. R. Role of sterols in membranes. Lipids. 1974 Aug;9(8):596–612. doi: 10.1007/BF02532509. [DOI] [PubMed] [Google Scholar]
  27. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. J Biol Chem. 1964 Jul;239:2379–2385. [PubMed] [Google Scholar]
  28. Porter T. D., Coon M. J. Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem. 1991 Jul 25;266(21):13469–13472. [PubMed] [Google Scholar]
  29. Shyadehi A. Z., Lamb D. C., Kelly S. L., Kelly D. E., Schunck W. H., Wright J. N., Corina D., Akhtar M. The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14 alpha-demethylase of Candida albicans (other names are: lanosterol 14 alpha-demethylase, P-45014DM, and CYP51). J Biol Chem. 1996 May 24;271(21):12445–12450. doi: 10.1074/jbc.271.21.12445. [DOI] [PubMed] [Google Scholar]
  30. Taton M., Rahier A. Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 alpha-demethylase from maize (Zea mays) seedlings. Biochem J. 1991 Jul 15;277(Pt 2):483–492. doi: 10.1042/bj2770483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trzaskos J. M., Bowen W. D., Shafiee A., Fischer R. T., Gaylor J. L. Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation. J Biol Chem. 1984 Nov 10;259(21):13402–13412. [PubMed] [Google Scholar]
  32. White T. C., Marr K. A., Bowden R. A. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev. 1998 Apr;11(2):382–402. doi: 10.1128/cmr.11.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES