Skip to main content
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1997 Apr;41(4):776–780. doi: 10.1128/aac.41.4.776

Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.

K Venkateswarlu 1, D E Kelly 1, S L Kelly 1
PMCID: PMC163793  PMID: 9087488

Abstract

Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

Full Text

The Full Text of this article is available as a PDF (205.8 KB).

Selected References

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

  1. Aoyama Y., Funae Y., Noshiro M., Horiuchi T., Yoshida Y. Occurrence of a P450 showing high homology to yeast lanosterol 14-demethylase (P450(14DM)) in the rat liver. Biochem Biophys Res Commun. 1994 Jun 30;201(3):1320–1326. doi: 10.1006/bbrc.1994.1848. [DOI] [PubMed] [Google Scholar]
  2. Aoyama Y., Yoshida Y., Sato R. Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. II. Lanosterol metabolism by purified P-450(14)DM and by intact microsomes. J Biol Chem. 1984 Feb 10;259(3):1661–1666. [PubMed] [Google Scholar]
  3. Barnes H. J., Arlotto M. P., Waterman M. R. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5597–5601. doi: 10.1073/pnas.88.13.5597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gonzalez F. J., Korzekwa K. R. Cytochromes P450 expression systems. Annu Rev Pharmacol Toxicol. 1995;35:369–390. doi: 10.1146/annurev.pa.35.040195.002101. [DOI] [PubMed] [Google Scholar]
  5. Hitchcock C. A., Dickinson K., Brown S. B., Evans E. G., Adams D. J. Purification and properties of cytochrome P-450-dependent 14 alpha-sterol demethylase from Candida albicans. Biochem J. 1989 Oct 15;263(2):573–579. doi: 10.1042/bj2630573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Kalb V. F., Loper J. C., Dey C. R., Woods C. W., Sutter T. R. Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene. 1986;45(3):237–245. doi: 10.1016/0378-1119(86)90021-1. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. King D. J., Azari M. R., Wiseman A. Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae. Xenobiotica. 1984 Jan-Feb;14(1-2):187–206. doi: 10.3109/00498258409151405. [DOI] [PubMed] [Google Scholar]
  11. Kirsch D. R., Lai M. H., O'Sullivan J. Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans. Gene. 1988 Sep 7;68(2):229–237. doi: 10.1016/0378-1119(88)90025-x. [DOI] [PubMed] [Google Scholar]
  12. Lai M. H., Kirsch D. R. Nucleotide sequence of cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans. Nucleic Acids Res. 1989 Jan 25;17(2):804–804. doi: 10.1093/nar/17.2.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miller W. L. Molecular biology of steroid hormone synthesis. Endocr Rev. 1988 Aug;9(3):295–318. doi: 10.1210/edrv-9-3-295. [DOI] [PubMed] [Google Scholar]
  14. Nelson D. R., Koymans L., Kamataki T., Stegeman J. J., Feyereisen R., Waxman D. J., Waterman M. R., Gotoh O., Coon M. J., Estabrook R. W. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics. 1996 Feb;6(1):1–42. doi: 10.1097/00008571-199602000-00002. [DOI] [PubMed] [Google Scholar]
  15. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  16. Shet M. S., Fisher C. W., Arlotto M. P., Shackleton C. H., Holmans P. L., Martin-Wixtrom C. A., Saeki Y., Estabrook R. W. Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase. Arch Biochem Biophys. 1994 Jun;311(2):402–417. doi: 10.1006/abbi.1994.1255. [DOI] [PubMed] [Google Scholar]
  17. Shet M. S., Fisher C. W., Holmans P. L., Estabrook R. W. Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11748–11752. doi: 10.1073/pnas.90.24.11748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Sloane D. L., So O. Y., Leung R., Scarafia L. E., Saldou N., Jarnagin K., Swinney D. C. Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase. Gene. 1995 Aug 19;161(2):243–248. doi: 10.1016/0378-1119(95)00211-n. [DOI] [PubMed] [Google Scholar]
  20. Sono H., Sonoda Y., Sato Y. Purification and characterization of cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from pig liver microsomes. Biochim Biophys Acta. 1991 Jul 12;1078(3):388–394. doi: 10.1016/0167-4838(91)90161-r. [DOI] [PubMed] [Google Scholar]
  21. Sonoda Y., Endo M., Ishida K., Sato Y., Fukusen N., Fukuhara M. Purification of a human cytochrome P-450 isozyme catalyzing lanosterol 14 alpha-demethylation. Biochim Biophys Acta. 1993 Sep 29;1170(1):92–97. [PubMed] [Google Scholar]
  22. Strömstedt M., Rozman D., Waterman M. R. The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterols. Arch Biochem Biophys. 1996 May 1;329(1):73–81. doi: 10.1006/abbi.1996.0193. [DOI] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Trzaskos J., Kawata S., Gaylor J. L. Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes. J Biol Chem. 1986 Nov 5;261(31):14651–14657. [PubMed] [Google Scholar]
  25. Venkateswarlu K., Denning D. W., Manning N. J., Kelly S. L. Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays. Antimicrob Agents Chemother. 1996 Jun;40(6):1382–1386. doi: 10.1128/aac.40.6.1382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Vermilion J. L., Coon M. J. Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. J Biol Chem. 1978 Apr 25;253(8):2694–2704. [PubMed] [Google Scholar]
  27. Waterman M. R., Jenkins C. M., Pikuleva I. Genetically engineered bacterial cells and applications. Toxicol Lett. 1995 Dec;82-83:807–813. doi: 10.1016/0378-4274(95)03597-4. [DOI] [PubMed] [Google Scholar]
  28. Yoshida Y., Aoyama Y. Interaction of azole antifungal agents with cytochrome P-45014DM purified from Saccharomyces cerevisiae microsomes. Biochem Pharmacol. 1987 Jan 15;36(2):229–235. doi: 10.1016/0006-2952(87)90694-0. [DOI] [PubMed] [Google Scholar]
  29. Yoshida Y., Aoyama Y. Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. I. Purification and spectral properties. J Biol Chem. 1984 Feb 10;259(3):1655–1660. [PubMed] [Google Scholar]
  30. van Nistelrooy J. G., van den Brink J. M., van Kan J. A., van Gorcom R. F., de Waard M. A. Isolation and molecular characterisation of the gene encoding eburicol 14 alpha-demethylase (cYP51) from Penicillium italicum. Mol Gen Genet. 1996 Apr 10;250(6):725–733. doi: 10.1007/BF02172984. [DOI] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES