Structural Requirements for Sterol 14alpha-Demethylases

描述（由申请人提供）：CYP51 催化 3 步 14β-去甲基化反应，该反应在甾醇生物合成中至关重要，是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）和钝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，也在低等真核生物和一些细菌中发现。我们已经确定了 CYP51 的第一个高分辨率结构，CYP51 是结核分枝杆菌 (MT) 的可溶形式。本次拨款申请的目标是利用该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。这是新应用程序的修订版。目标 1 - 来自不同门的 50 多个 CYP51 序列的比对显示大约 40 个氨基酸完全或非常高度保守。我们建议保守残基是 14β-去甲基酶的最低结构要求。 MT 和人 CYP51 的定点诱变将允许分析每个残基的作用。将研究每个突变体的细菌表达水平、底物结合光谱分析和催化活性。底物诱导的构象变化的 X 射线结构、荧光和圆二色性分析以及底物结合的停流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 仅代谢单一底物（obtusifoliol - 在 C4 处仅具有单个甲基的生物 CYP51 底物），而来自其他门的形式代谢多种甾醇。植物中严格保守的第二组残基的定点诱变将用于根据上述方法确定植物CYP51底物特异性的结构基础。目标 3 - 用唑类 CYP51 抑制剂治疗致病性白色念珠菌感染，导致某些耐药念珠菌菌株中出现 CYP51 突变形式。这些突变形式在单个 CYP51 活性位点上与唑类抑制剂的结合较差，而与底物的结合则正常。对 MTCYP51 中这些突变的生物物理特性的研究将为病原体中 CYP51 耐药性提供解释。这些目标共同将为 CYP51 的结构/功能提供新的、广泛的见解，这是对广泛分布的重要 CYP 家族进行详细分析的第一次机会。此外，我们预计这些研究将提供有关 P450 结构/功能的一般信息，并为治疗病原体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.