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Antimicrobial Agents and Chemotherapy, July 1999, p. 1725-1728, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Delta 22-Desaturase from the Pathogenic Fungus Candida glabrata

David C. Lamb,1 Segula Maspahy,1 Diane E. Kelly,1 Nigel J. Manning,2 Antonia Geber,3 John E. Bennett,3 and Steven L. Kelly1,*

Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales SY23 3DA,1 and Chemical Pathology, Sheffield Childrens Hospital, Sheffield, England S10 2UH,2 United Kingdom, and Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 208923

Received 19 October 1998/Returned for modification 28 January 1999/Accepted 4 May 1999

Sterol Delta 22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14alpha -demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Delta 22-desaturase activity in a reconstituted system with NADPH-cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 µM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Delta 22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Delta 22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.

* Corresponding author. Mailing address: Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales SY23 3DA, United Kingdom. Phone: 44 (1970) 621515. Fax: 44 (1970) 622350. E-mail: StevenKelly{at}aber.ac.uk.

Antimicrobial Agents and Chemotherapy, July 1999, p. 1725-1728, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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