ebericol and obtusifoliol

Metabolism of lanosterol (LAN), 24-methylene-24,25-dihydrolanosterol (24-methyleneDHL), dihydrolanosterol (DHL) and obtusifoliol (OBT) by purified human, plant (Sorghum bicolor) and fungal (Candida albicans) sterol 14alpha-demethylase (CYP51; P450(14DM)) reconstituted with NADPH cytochrome P450 reductases was studied in order to elucidate the substrate specificity and sterol stereo- and regio-structural requirements for optimal CYP51 activity. Both human and C. albicans CYP51 could catalyse 14alpha-demethylation of each substrate with varying levels of activity, but having slightly higher activity for their respective endogenous substrates in vivo, dihydrolanosterol for human CYP51 (Vmax = 0.5 nmol/min/nmol CYP51) and 24-methylene-24,25-dihydrolanosterol for C. albicans CYP51 (Vmax = 0.3 nmol/min/nmol CYP51). In contrast, S. bicolor CYP51 showed strict substrate specificity and selectivity towards its own endogenous substrate, obtusifoliol (Vmax = 5.5 nmol/min/nmol CYP51) and was inactive towards 14alpha-demethylation of lanosterol, 24-methylene-24,25-dihydrolanosterol and dihydrolanosterol. These findings confirm that the presence of the 4beta-methyl group in the sterol molecule renders the plant CYP51 incapable of 14alpha-demethylation thus revealing the strict active site conservation of plant CYP51 during evolution.

Topics: Candida albicans; Cholestadienols; Cytochrome P-450 Enzyme System; Edible Grain; Humans; Lanosterol; Molecular Structure; Oxidoreductases; Sterol 14-Demethylase; Substrate Specificity

Mucosal candidosis is an almost inevitable consequence of AIDS. Resistance to fluconazole therapy associated with enhanced tolerance, detectable in microbiological estimation of sensitivity, occurs in up to 10% of cases with late-stage AIDS. We report here our biochemical analysis of the basis of resistance in a study of two susceptible and two resistant isolates. Resistance was not associated with a change in the target enzyme sterol 14 alpha-demethylase, as indicated by equivalent levels of fluconazole inhibition of activity in extracts from all four isolates, or by mutations in sterol delta desaturase as previously observed in Saccharomyces cerevisiae and Ustilago maydis. Reduced cellular content of fluconazole in the resistant isolates of between six to ten-fold was observed which could account for their resistant phenotype.

Topics: AIDS-Related Opportunistic Infections; Antifungal Agents; Candida albicans; Candidiasis, Chronic Mucocutaneous; Cholestadienols; Cytochrome P-450 Enzyme System; Drug Resistance, Microbial; Ergosterol; Fluconazole; Humans; Lanosterol; Microbial Sensitivity Tests; Oxidoreductases; Sterol 14-Demethylase

Two classes of polyene-resistant mutants were isolated from survivors of N-methyl-N'-nitro-N-nitrosoguanidine treatment of a wild-type Candida albicans. An analysis of the major sterols of one class revealed an accumulation of lichesterol and fecosterol while the other class accumulated eburicol, obtusifoliol, and lanosterol with minor quantities of C28 sterols.

Topics: Amphotericin B; Candicidin; Candida albicans; Cholestadienols; Drug Resistance, Microbial; Ergosterol; Lanosterol; Mutation; Natamycin; Nystatin; Phytosterols; Triterpenes