Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Aouatef Bellamine, Galina L. Lepesheva, and Michael R. Waterman

Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-0146

Corresponding Author: aouatef.bellamine{at}vanderbilt.edu

14a-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists and some bacteria) catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-direct mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms, human, fungal and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding whereas the F145L mutant results in 5-fold increase in its IC50 for fluconazole, suggesting that F145 (conserved only in fungal 14a-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi providing a basis for treatment of fungal infections. While the C. albicans Y132H and human Y145H CYP51 mutants demonstrate essentially no effect on substrate metabolism, the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Since these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasizes that fluconazole and substrate binding are uncoupled properties

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