The R467K amino acid substitution in Candida albicans sterol 14 alpha-demethylase causes drug resistance through reduced affinity

The cytochrome P450 sterol 14 alpha-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the tar get for azole antifungal compounds. We have undertaken site-directed mutati on of C. albicans CYP51 to produce a recombinant mutant protein with the am ino 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 t he affinity of the enzyme for fluconazole, as shown by ligand binding studi es. The specific activity of CYP51(R467K) for the release of formic acid fr om 3 beta-[32-H-3]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 fr actions 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 resistanc e observed clinically can result from the altered azole affinity of the fun gal CYP51 enzyme.