MECHANISMS INVOLVED IN FLAVIN LINKED OXYGEN METABOLISM

面临着在有氧环境中生长的生化困境 环境，但没有能力合成细胞色素或 其他的血红蛋白，肠球菌依赖于几种非常不寻常的 黄素蛋白；这些酶在氧气依赖中起着关键作用 能量代谢和细胞对氧化应激的防御。 NADH氧化酶(O2和GT；H2O)和NADH过氧化物酶(H2 02和GT；2H20)也 代表了一种新型的黄素蛋白，其中第二个非黄素 氧化还原中心被鉴定为稳定的半胱氨酸-磺酸 (CyS-SOH)。这项研究的一个目标是完成结晶学 天然Cys-SOH和Cys-SH形式的过氧化物酶分析 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 对NADH氧化酶进行了分析，重点阐述了四种酶的作用机制。 参与O2和GT；2H2O还原的电子转移将是 正在追查；初步证据表明，黄素-C(4a)- 可能会形成过氧化氢物种。三聚氰胺的结晶学研究 Cys42Ser突变氧化酶将提供有关 区分氧化物酶末端过氧化物酶的结构基础 这两个同源蛋白质中的催化循环。此外，这一点 结构工作将扩大到包括来自 化脓性链球菌和肺炎链球菌，尤其是最近 遗传证据表明，这种酶可能是一种有希望的靶标 在这些重要的人类病原体中基于结构的药物设计。这个 肠球菌对甘油的摄取和分解代谢需要 两种胞浆酶的协同作用：甘油激酶(GlpK)和 依赖于FAD的α-甘油磷酸酶(GlpO)。数列 对GlpO的分析令人惊讶地表明，这种胞浆酶是 与其他依赖FAD的氧化酶(O2和GT；H_2O_2)无关，而与 膜相关α-甘油磷酸脱氢酶(GlpDS)； 计划进行GlpO的结构和动力学研究，这也应该 提供有关两种底物的决定因素的详细见解 同源GlpD的结合和亚细胞定位。

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.