+ Site Statistics
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51)

Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51)

Antimicrobial Agents and ChemoTherapy 54(10): 4235-4245

Purified Candida albicans sterol 14-α demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K(s) values of 11 and 25 μM, respectively, and a K(m) value of 6 μM for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (ΔA(max)) and the azole concentration required to obtain a half-ΔA(max) being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K(d) values of 10 to 26 μM under oxidative conditions, compared with 47 μM for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min(-1)), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.

(PDF emailed within 0-6 h: $19.90)

Accession: 051738941

Download citation: RISBibTeXText

PMID: 20625155

DOI: 10.1128/AAC.00587-10

Related references

Cytochrome P-450-dependent 14 alpha -sterol demethylase of Candida albicans and its interaction with azole antifungals. Biochemical Society Transactions 19(3): 782-787, 1991

Inhibition of 14 alpha-sterol demethylase activity in Candida albicans Darlington does not correlate with resistance to azole. Journal of Medical and Veterinary Mycology 25(5): 329-333, 1987

Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans. Biochemical Journal 266(2): 475-480, 1990

Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450. Antimicrobial Agents and ChemoTherapy 43(5): 1163-1169, 1999

Identification of y118 amino acid residue in Candida albicans sterol 14 alpha-demethylase associated with the enzyme activity and selective antifungal activity of azole analogues. Biological and Pharmaceutical Bulletin 30(7): 1246-1253, 2007

Purification and properties of cytochrome P-450-dependent 14 alpha-sterol demethylase from Candida albicans. Biochemical Journal 263(2): 573-579, 1989

Azole affinity of sterol 14α-demethylase (CYP51) enzymes from Candida albicans and Homo sapiens. Antimicrobial Agents and ChemoTherapy 57(3): 1352-1360, 2013

Azole Affinity of Sterol 14alpha-Demethylase CYP51 Enzymes from Candida albicans and Homo sapiens. 2013

Structural analyses of iCandida albicans sterol 14α-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis. Journal of Biological Chemistry 292(16): 6728-6743, 2017

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). Journal of Biological Chemistry 271(21): 12445-12450, 1996

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds. Journal of Biochemistry 137(5): 625-632, 2005

Non-azole inhibitors of lanosterol 14-alpha-demethylase in Candida albicans CCH442. Abstracts of the Interscience Conference on Antimicrobial Agents & Chemotherapy 35(0): 131, 1995

A non-azole inhibitor of lanosterol 14 alpha-methyl demethylase in Candida albicans. Journal of Antimicrobial ChemoTherapy 30(6): 781-790, 1992

Identification of Y118 Amino Acid Residue in Candida albicans Sterol 14α-Demethylase Associated with the Enzyme Activity and Selective Antifungal Activity of Azole Analogues. Biological & Pharmaceutical Bulletin 30(7): 1246-1253, 2007

Sterol 14 alpha -demethylase and its inhibition: structural considerations on interaction of azole antifungal agents with lanosterol 14 alpha -demethylase (P-45014DM) of yeast. Biochemical Society Transactions 19(3): 778-782, 1991