The mutation T315A in Candida albicans sterol 14 alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity
The mutation T315A in Candida albicans sterol 14 alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity
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DOI：
10.1074/jbc.272.9.5682
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发表时间：
1997-02-28
影响因子：
4.8
通讯作者：
Kelly, SL
中科院分区：
文献类型：
--
作者：
Lamb, DC;Kelly, DE;Kelly, SL
甾醇14α-脱甲基酶(P45051)是唑类抗真菌化合物的靶标，而对这些药物和农用化学品的抗药性具有重要的实际意义，我们对在酿酒酵母60中异源表达的白色念珠菌P45051进行了定点突变，T315A改变了酶的模型结构，使酶活性降低了2倍，如预测的那样，去除了与甾醇底物的3-OH形成氢键的残基，并帮助将其定位在活性部位，这种改变扰乱了血红素环境，导致一氧化碳差谱发生改变，最大值达445mN。这些变化还降低了该酶对唑类抗真菌药酮康唑和氟康唑的亲和力，并在半乳糖诱导表达后，导致酿酒酵母S转化子对唑类药物的抗性增加了45倍。这是第一个通过降低唑类亲和力而使靶标酶发生单一碱基改变而导致对唑类药物产生抗药性的例子。
Sterol 14 alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance, We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae 60 probe a model structure for the enzyme, The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site, This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 mn. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 45-fold azole resistance in transformants of S, cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.