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标题：Identification of Y118 Amino Acid Residue in Candida albicans Sterol 14α-Demethylase Associated with the Enzyme Activity and Selective Antifungal Activity of Azole Analogues
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作者：Shuang-Hong Chen ; Chun-Quan Sheng ; Xiao-Hui Xu 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2007
卷号：30
期号：7
页码：1246-1253
DOI：10.1248/bpb.30.1246
出版社：The Pharmaceutical Society of Japan
摘要：Our previous publication established a model to predict that the phenyl group of the C-3 side chain of azole antifungal compounds interacts with the phenol group of Tyr118 through the formation of π–π face-to-edge interaction. To verify this prediction, wild type and three site-directed mutants of the Y118 residue of Candida albicans sterol 14α-demethylase P450 (CACYP51) were constructed and heterologously expressed in Saccharomyces cerevisiae with deletion of the CYP51 gene. With the strains obtained and microsome enzymes separated, cell susceptibility and CACYP51 activity were examined with the 5 novel azole compounds based on the molecular modeling in comparison with fluconazole. After alteration of Y118 with Y118A , Y118F , and Y118T by a single base substitution, the expression levels of CACYP51 protein were not affected. However, these mutations markedly decreased its catalytic activity respectively; the mutation changes also decreased azole susceptibility, indicating the structural importance of the Y118 residue in maintaining CACYP51 activity and in determining azole susceptibility. In addition, our synthetic compounds with the phenyl group side chain attached to C3 produced higher susceptibility against S. cerevisiae with expression of CACYP51 and exhibited more potent inhibitory effects on CACYP51 activity in comparison with fluconazole, suggesting that the phenyl group of C3 side chain substitutes is also important for selective binding to target enzymes.
关键词：Candida albicans;gene deletion;mutation;inhibitor binding;CYP51