Structural Requirements for Sterol 14alpha-Demethylases

描述(由申请人提供)：CYP51催化3步14？去甲基化反应，这是甾醇生物合成中必不可少的，是将羊毛甾醇转化为胆固醇(动物)、24-亚甲基二氢羊毛甾醇转化为麦角甾醇(真菌/酵母)和长叶醇转化为植物甾醇(植物)所必需的。它是细胞色素P450超家族中分布最广泛的成员，也存在于低等真核生物和一些细菌中。我们已经确定了来自结核分枝杆菌(MT)的可溶性形式的CYP51的第一个高分辨结构。这项资助申请的目的是利用这一结构信息和相对大量的已知CYP51序列来提供对CYP51结构/功能的详细了解。这是一个新应用程序的修订版。目的1对来自不同门的50多个CYP51序列进行比对，结果显示约40个氨基酸完全或高度保守。我们建议保守残基是14？-脱甲基酶的最低结构要求。MT和人类细胞色素P51的定点突变将允许分析每一个残基的作用。细菌的表达水平，底物结合的光谱分析和催化活性将在每个突变体中进行研究。对底物诱导的构象变化的X射线结构、荧光和圆二色谱分析，以及底物结合的停止流动分析也将提供所选突变的生物物理细节。目的植物中的2-细胞色素P51只代谢一种底物--长叶醇--一种生物底物，其C4上只有一个甲基。根据上面提到的方法，对第二组在植物中严格保守的残基进行定点突变将被用来确定植物CYP51底物特异性的结构基础。目的用唑类细胞色素P5 1抑制剂治疗致病性白念珠菌感染，可导致部分耐药株的细胞色素P5 1基因突变。这些突变形式与唑类抑制剂的结合效果较差，而与底物的结合正常，两者都位于单一的CYP51活性部位。对这些突变的生物物理性质的研究将为病原菌对CYP51的耐药性提供解释。这些目标将为我们提供对CyP51结构/功能的新的和广泛的洞察力，这将是对一个广泛分布和基本的CYP家族进行如此详细分析的第一次机会。此外，我们预计这些研究将导致关于P450结构/功能的一般信息，并为治疗病原性感染的药物设计提供有用的见解。

DESCRIPTION (provided by applicant): CYP51 catalyzes the 3-step 14?-demethylation reaction that is essential in sterol biosynthesis, being required for conversion of lanosterol to cholesterol (animals), 24-methylenedihydrolanosterol to ergosterol (fungi/yeast) and obtusifoliol to phytosterols (plants). It is the most widely distributed member of the cytochrome P450 superfamily, also being found in lower eukaryotes and some bacteria. We have determined the first high resolution structure of a CYP51, the soluble form from Mycobacterium tuberculosis (MT). The goal of this grant application is to take advantage of this structural information and the relatively large number of known CYP51 sequences to provide a detailed understanding of CYP51 structure/function. This is a revision of a new application. Aim 1 - Alignment of the more than 50 CYP51 sequences from different phyla shows about 40 amino acids completely or very highly conserved. We propose the conserved residues to be the minimal structural requirement for 14?-demethylases. Site-directed mutagenesis of MT and human CYP51 will permit analysis of the role of each of these residues. Levels of bacterial expression, spectral analysis of substrate binding and catalytic activity will be studied in each mutant. X-ray structure, fluorescence and circular dichroism analysis of substrate-induced conformational changes, and stopped flow analysis of substrate binding will also provide biophysical detail of selected mutations. Aim 2 - CYP51 from plants metabolizes only a single substrate (obtusifoliol--the biological CYP51 substrate having just a single methyl group at C4) while forms from other phyla metabolize multiple sterols. Site-directed mutagenesis of a second set of residues conserved strictly in plants will be used to determine the structural basis of plant CYP51 substrate specificity, relying on methods cited above. Aim 3 - Treatment of pathogenic Candida albicans infections with azole CYP51 inhibitors leads to mutant forms of CYP51 in some drug-resistant Candida strains. These mutant forms bind azole inhibitors less well while binding substrate normally, both in the single CYP51 active site. Investigation of biophysical properties of these mutations in MTCYP51 will provide an explanation for CYP51 drug resistance in pathogens. Together these aims will provide new and extensive insight into structure/function of CYP51, the first opportunity for such detailed analysis of a widely distributed and essential CYP family. In addition we expect that these studies will lead to general information about P450 structure/function and provide useful insight into drug design for treatment of pathogenic infections.