Structural Requirements for Sterol 14alpha-Demethylases

描述（由申请人提供）：CYP51催化3-步骤14？-去甲基化反应，在甾醇生物合成中是必需的，在羊毛甾醇转化为胆固醇（动物）、24-亚二氢羊毛甾醇转化为麦角甾醇（真菌/酵母）和烟油醇转化为植物甾醇（植物）中都需要。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CYP51的第一个高分辨率结构，来自结核分枝杆菌（MT）的可溶性形式。本资助申请的目标是利用这些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序列的比对显示，约有40个氨基酸完全保守或高度保守。我们提出保守残基是14?-去甲基化酶的最小结构要求。MT和人类CYP51的定点突变将允许分析这些残基的作用。细菌表达水平、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。底物诱导构象变化的x射线结构、荧光和圆二色性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。目标2 -来自植物的CYP51只代谢单一底物（烟叶油醇-生物CYP51底物只有一个甲基在C4），而来自其他门的形式代谢多种甾醇。依靠上述方法，对植物中严格保守的第二组残基进行定点诱变，以确定植物CYP51底物特异性的结构基础。目的3 -用唑类CYP51抑制剂治疗致病性白色念珠菌感染可导致一些耐药念珠菌株CYP51突变。这些突变形式与唑抑制剂结合较差，而与底物正常结合，两者都在单个CYP51活性位点。研究这些突变的生物物理特性将有助于解释CYP51在病原体中的耐药性。总之，这些目标将为CYP51的结构/功能提供新的和广泛的见解，这是第一次有机会对广泛分布和必需的CYP家族进行如此详细的分析。此外，我们期望这些研究将导致关于P450结构/功能的一般信息，并为治疗致病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.