Lamb D C, Kelly D E, Schunck W H, Shyadehi A Z, Akhtar M, Lowe D J, Baldwin B C, Kelly S L
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2UH, United Kingdom.
J Biol Chem. 1997 Feb 28;272(9):5682-8. doi: 10.1074/jbc.272.9.5682.
Sterol 14alpha-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 to 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 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-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.
甾醇14α-脱甲基酶（P45051）是唑类抗真菌化合物的作用靶点，对这些药物和农用化学品产生耐药性具有重要的实际意义。我们对在酿酒酵母中异源表达的白色念珠菌P45051进行了定点诱变，以探究该酶的模型结构。T315A突变使酶活性降低了2倍，这与去除与甾醇底物3-OH形成氢键的残基的预测结果相符，该残基有助于将甾醇定位在活性位点。这种改变扰乱了血红素环境，导致还原型一氧化碳差光谱发生改变，最大吸收峰位于445 nm。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，在半乳糖诱导表达后，导致酿酒酵母转化体产生4至5倍的唑类耐药性。这是目标酶中单个碱基变化通过降低唑类亲和力赋予唑类耐药性的首个实例。
