Structural Requirements for Sterol 14alpha-Demethylases

甾醇 14α-脱甲基酶的结构要求

• 批准号: 6732365
• 负责人: MICHAEL R WATERMAN
• 金额: $ 28.99万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6732365
• 关键词: Candida albicans; X ray crystallography; active sites; antibacterial agents; antifungal agents; bacterial disease; circular dichroism; conformation; cytochrome P450; drug design /synthesis /production; drug resistance; enzyme activity; enzyme inhibitors; fluorescence resonance energy transfer; fluorescence spectrometry; infection; mycosis; protein structure function; site directed mutagenesis; sterols; stop flow technique

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.

描述（申请人提供）：CYP 51催化3步14？-脱甲基反应是甾醇生物合成中必不可少的，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。我们已经确定了第一个高分辨率结构的CYP 51，可溶性形式从结核分枝杆菌（MT）。这项资助申请的目的是利用这些结构信息和相对大量的已知CYP 51序列，以提供对CYP 51结构/功能的详细了解。这是新应用程序的修订版。目的1 -对来自不同门的50多个CYP 51序列进行比对，发现约40个氨基酸完全或高度保守。我们建议保守的残基是14？-脱甲基酶MT和人CYP 51的定点突变将允许分析这些残基中的每一个的作用。将在每种突变体中研究细菌表达水平、底物结合的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。目的2 -来自植物的CYP 51仅代谢单一底物（obtusifoliol-在C4处仅具有单个甲基的生物CYP 51底物），而来自其他门的形式代谢多种甾醇。依赖于上面引用的方法，在植物中严格保守的第二组残基的定点诱变将用于确定植物CYP 51底物特异性的结构基础。目的3 -用唑类CYP 51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CYP 51的突变形式。这些突变形式结合唑类抑制剂的能力较差，而结合底物的能力正常，两者都在单个CYP 51活性位点。对MTCYP 51中这些突变的生物物理特性的研究将为病原体中CYP 51耐药性提供解释。这些目标将为CYP 51的结构/功能提供新的和广泛的见解，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导致有关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。