Cholesterol Oxidase Loop Residues and Catalysis

说明(申请人提供)：胆固醇氧化酶是一种黄素酶，催化胆固醇氧化和异构化为胆甾烯-3-酮。胆固醇氧化酶在商业上用于测定血清胆固醇浓度，具有杀虫作用，并用于研究膜结构。最近的证据表明，胆固醇氧化酶可能参与了一些微生物的发病机制，如马红球菌和结核分枝杆菌。建议的研究将为活性所需的脂类和黄素氧化途径提供精确的分子模型。这些模型对于胆固醇氧化酶商品化的发展、作为监测脂筏的细胞膜的有用和可靠的工具、研究胆固醇氧化酶在细菌发病机制中的作用以及开发抗菌抑制剂都是重要的。要检验的具体假设是：1)胆固醇氧化酶可以区分同一膜上不同组成的含胆固醇脂类结构域，即液体无序结构域和液体有序结构域；2)在催化过程中，该酶利用一个大的界面与脂质双层结合；以及3)溶剂和活性部位的黄素之间的一条门控隧道控制氧的访问。

DESCRIPTION (provided by applicant): Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Cholesterol oxidase is used commercially for the determination of serum cholesterol concentrations, is insecticidal, and is used to study membrane structure. Recent evidence suggests that cholesterol oxidase may be involved in the pathogenesis of some microorganisms such as Rhodococcus equi and Mycobacterium tuberculosis. The studies proposed will provide a precise molecular model of the lipid requirements for activity and of the pathway to flavin oxidation. These models are important for the development of cholesterol oxidase as a commercial product, as a useful and reliable tool in the study of cellular membranes for monitoring lipid rafts, for investigating the role of cholesterol oxidase in bacterial pathogenesis, and for developing anti-bacterial inhibitors. The specific hypotheses that will be tested are that 1) cholesterol oxidase can distinguish between cholesterol-containing lipid domains of different composition in the same membrane, i.e., between liquid-disordered domains and liquid-ordered domains (rafts); 2) the enzyme employs a large interface to associate with the lipid bilayer during catalysis; and 3) a gated tunnel between the solvent and the flavin at the active site controls oxygen access.