MECHANISMS INVOLVED IN FLAVIN LINKED OXYGEN METABOLISM

在有氧环境中生长的生物化学难题 环境，但没有能力合成细胞色素或 其他血红素蛋白，肠球菌依赖于几个非常不寻常的 黄素蛋白;这些酶在氧依赖性 能量代谢和细胞防御氧化应激。 NADH氧化酶（O2>H2O）和NADH过氧化物酶（H2 O2> 2 H2O）也 代表了一种新型的黄素蛋白，其中第二种非黄素 氧化还原中心已被确定为稳定的半胱氨酸-次磺酸 （Cys-SOH）。这项研究的一个目标是完成晶体学 分析过氧化物酶的天然Cys-SOH和Cys-SH形式， 高分辨率;关于Cys-SOH模式的信息 过氧化物酶内的稳定也可以应用于许多 氧化还原调节的DNA结合蛋白，如OxyR，Cys- SOH/Cys-SH氧化还原中心已被提出。 停流动力学 NADH氧化酶的分析，集中在四个机制， 电子转移参与O2> 2 H2O的还原，将是 追求;初步证据表明，黄素-C（4a）- 可形成氢过氧化物物质。的晶体学研究 Cys 42 Ser突变体氧化酶将提供关于 区分氧化酶末端过氧化物酶的结构基础 这两种同源蛋白质的催化循环。 另外这款 结构工作将扩大到包括NADH氧化酶， 化脓性链球菌和S.肺炎，特别是鉴于最近 遗传证据表明，这种酶可能是一个有希望的目标， 基于结构的药物设计在这些重要的人类病原体。的 肠球菌对甘油的摄取和催化需要 两种胞浆酶的协同作用：甘油激酶（GlpK）和 FAD依赖性α-甘油磷酸氧化酶（GlpO）。序列 GlpO的分析令人惊讶地表明，这种胞质酶是 与其他FAD依赖性氧化酶（O2> H2 O2）无关，但与 膜相关α-甘油磷酸脱氢酶（GlpDs）; 计划对GlpO进行结构和动力学研究， 提供关于两种底物的决定因素的详细见解 结合和亚细胞定位在同源GlpDs。

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.