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Antimicrobial Agents and Chemotherapy, January 2000, p. 63-67, Vol. 44, No. 1
0066-4804/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The R467K Amino Acid Substitution in Candida albicans Sterol 14alpha -Demethylase Causes Drug Resistance through Reduced Affinity

David C. Lamb,1 Diane E. Kelly,1 Theodore C. White,2 and Steven L. Kelly1,*

Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, United Kingdom,1 and Department of Pathobiology, School of Public Health and Community Medicine, University of Washington and Seattle Biomedical Research Institute, Seattle, Washington 981092

Received 19 March 1999/Returned for modification 5 August 1999/Accepted 14 October 1999

The cytochrome P450 sterol 14alpha -demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3beta -[32-3H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.

* Corresponding author. Mailing address: Institute of Biological Sciences, Edward Llwyd Bldg., University of Wales Aberystwyth, Aberystwyth SY23 3DA, United Kingdom. Phone: 01970 621515. Fax: 01970 622350. E-mail: Steven.Kelly{at}aber.ac.uk.

Antimicrobial Agents and Chemotherapy, January 2000, p. 63-67, Vol. 44, No. 1
0066-4804/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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