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Human and Candida albicans CYP51 were purified to homogeneity after GAL10-based heterologous expression in yeast in order to resolve the basis for the selective inhibition of the fungal enzyme over the human orthologue by the azole drugs ketoconazole and itraconazole, used in the treatment of systemic fungal infection. The purified proteins have similar spectral characteristics, both giving a maximum at 448 nm in reduced carbon monoxide difference spectra. Substrate affinity constants of 20.8 and 29.4 microM and Vmax of 0. 15 and 0.47 nmol/min/nmol were observed for C. albicans and human enzymes, respectively, in reconstituted enzymatic assays, using an intermediate of the demethylation reaction [32-3H]-3beta-hydroxylanost-7-en-32-ol as the substrate. Both enzymes gave similar type II spectra on titration with drugs, but a reduced affinity was observed for human CYP51 using the ability of carbon monoxide to displace the drug as a ligand and by calculation of IC50. However, although the results indicate higher affinity of the drugs for their target CYP51 in the major fungal pathogen C. albicans, when compared directly to CYP51 from humans, the difference was less than 10-fold. This difference is an order of magnitude lower than previously reported data based on measurements using unpurified human CYP51 enzyme preparations. Consequently, increased azole doses to combat resistant candidaemia may well inhibit endogenous human CYP51 and the potential consequences are discussed.
Copyright 1999 John Wiley & Sons, Ltd.