MECHANISMS INVOLVED IN FLAVIN LINKED OXYGEN METABOLISM

黄素相关氧代谢的机制

• 批准号: 2398114
• 负责人: AL CLAIBORNE
• 金额: $ 30.18万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2398114
• 关键词: DNA binding protein; Enterococcus; NAD(P)H dehydrogenase; Streptococcus mutans; Streptococcus pneumoniae; Streptococcus pyogenes; X ray crystallography; active sites; chemical kinetics; crystallization; cysteine; enzyme mechanism; enzyme structure; flavin adenine dinucleotide; flavoproteins; glycerol 3 phosphate dehydrogenase; glycerol kinase; oxidation reduction reaction; peroxidases; recombinant proteins; stop flow technique

Presented with the biochemical dilemma of growing in an aerobic environment, but without the capacity to synthesize cytochromes or other hemeproteins, the enterococci depend on several very unusual flavoproteins; these enzymes play key roles both in oxygen-dependent energy metabolism and in the cellular defense against oxidative stress. The NADH oxidase (O2>H2O) and NADH peroxidase (H2 02>2H20) also represent a new type of flavoprotein in which the second non-flavin redox center has been identified as a stable cysteine-sulfenic acid (Cys-SOH). One goal of this study is to complete the crystallographic analysis of the native Cys-SOH and Cys-SH forms of the peroxidase at high resolution; information concerning the modes of Cys-SOH stabilization within the peroxidase can also be applied to a number of redox-regulated DNA-binding proteins, such as OxyR, for which Cys- SOH/Cys-SH redox centers have been proposed. Stopped-flow kinetic analyses of the NADH oxidase, focused on the mechanism of the four- electron transfer involved in the reduction of O2>2H2O, will be pursued; preliminary evidence indicates that a flavin-C(4a)- hydroperoxide species may be formed. Crystallographic studies of the Cys42Ser mutant oxidase will provide detailed information on the structural basis for the distinction between oxidase end peroxidase catalytic cycles in these two homologous proteins. In addition, this structural work will be expanded to include the NADH oxidases from Streptococcus pyogenes and S. pneumoniae, especially in view of recent genetic evidence that this enzyme may represent a promising target for structure-based drug design in these important human pathogens. The uptake and catabolism of glycerol in the enterococci requires the concerted actions of two cytosolic enzymes: glycerol kinase (GlpK) and the FAD-dependent alpha-glycerophosphate oxidase (GlpO). Sequence analysis of GlpO indicates surprisingly that this cytosolic enzyme is not related to other FAD-dependent oxidases (O2>H2O2) but to the membrane-associated alpha-glycerophosphate dehydrogenases (GlpDs); structural and kinetic studies of GlpO are planned which should also provide detailed insight regarding the determinants of both substrate binding and subcellular localization in the homologous GlpDs.

出现有氧生长的生化困境 环境，但没有能力合成细胞色素或 其他血蛋白，肠球菌取决于几个非常不寻常的 黄素蛋白；这些酶在氧气依赖性中都起关键作用 能量代谢和抗氧化应激的细胞防御。 NADH氧化酶（O2> H2O）和NADH过氧化物酶（H2 02> 2H20）也 代表一种新型的黄蛋白 氧化还原中心已被确定为稳定的半胱氨酸硫酸 （cys-soh）。这项研究的目标之一是完成晶体学 分析过氧化物酶的天然CYS-SOH和CYS-SH形式 高分辨率;有关cys-soh模式的信息 过氧化物酶内的稳定也可以应用于多个 氧化还原调节的DNA结合蛋白，例如oxyr，其中cys- 已经提出了SOH/CYS-SH氧化还原中心。 停止流动动力学 NADH氧化酶的分析，侧重于四氧化酶 涉及减少O2> 2H2O的电子转移将为 追捕；初步证据表明，黄素-C（4A） - 可以形成氢过氧化物。晶体学研究 cys42ser突变氧化酶将提供有关该氧化的详细信息 氧化酶末端过氧化物酶之间区别的结构基础 这两个同源蛋白质中的催化循环。 此外，这 结构性工作将扩展到包括来自的NADH氧化酶 链球菌为链球菌和肺炎链球菌，特别是鉴于最近 遗传证据表明该酶可能代表了 这些重要的人类病原体中基于结构的药物设计。这 肠球菌中甘油的摄取和分解代谢需要 两种胞质酶的协同作用：甘油激酶（GLPK）和 FAD依赖性α-甘油磷酸氧化酶（GLPO）。顺序 GLPO的分析表明该胞质酶是 与其他依赖FAD的氧化酶（O2> H2O2）无关，而与 膜相关的α-甘油磷酸脱氢酶（GLPD）； 计划对GLPO进行结构和动力学研究，还应 提供有关两种底物决定因素的详细见解 同源GLPD中的结合和亚细胞定位。