Mechanisms Involved in Flavin-linked Oxygen Metabolism

描述：（由申请人提供）已知乳酸菌 代表了氧代谢中生物化学多样性的强有力范例， 这是由于它们缺乏呼吸细胞色素和其他血红素蛋白。相反 这些生物体依赖于不寻常的黄素连接的呼吸酶的网络， 这项申请的重点是详细的结构和机制研究 这一组中有三个特别有趣的代表：（1）NADH 来自严重人类病原体的氧化酶（Nox; 2NADH + O2 to 2NAD++2H2O） 化脓性链球菌，（2）NADH过氧化物酶（Npx; NADH + H2 O2-至NAD++2H2O） （3）α-甘油磷酸氧化酶（GlpO; α-甘油磷酸盐+ O2 -至二羟丙酮磷酸盐+H2 O2）， GlpO序列与链球菌的GlpO序列密切相关。 膜相关α-甘油磷酸脱氢酶（GlpDs）;这些酶是 其主要特征在于存在GlpO特有的50个残基插入物， 其在有限的蛋白水解实验中进一步显示， 代表了一个在黄素化过程中起关键作用的柔性表面区域， 还原GlpO结构的阐明应该提供对GlpO的结构的深入了解。 相关政府间机构的结构，并应更好地界定 GlpO表面在甘油磷酸盐氧化过程中形成环。而结构 Nox和Npx之间同源性已经用新晶体显著增强 在当前项目期间，只有最近才有第三名成员 在这一小类NAD（P）H-连接的过氧化物/二硫键还原酶中， FAD依赖性辅酶A-二硫化物还原酶（CoADR; NADPH +） CoASSCoA到NADP++2CoASH）的分离 金黄色。CoADR很好地补充了这组新兴的黄素蛋白， 利用单个活性位点Cys作为基本氧化还原中心的基础- 用Nox和Npx记录的Cys-次磺酸（Cys-SOH），以及Cys-SSCoA CoADR中存在二硫化物。另一个长期目标是 应用是结构，氧化还原和机械的持续定义 Nox和Npx中Cys-SOH功能的参数，因为这些酶提供了 蛋白质-SOH中心现在已知参与各种 氧化还原信号和催化方面进行了分析。新结构 Nox和Npx的可用数据也为继续分析 O2还原为2 H2O的相对罕见的功能区别 Nox的催化作用和Npx的过氧化反应。可解释 最近计算的CoADR电子密度图现在使得有可能 模型和完善这种酶的原子结构，从而允许 与Nox和Npx的详细比较，并提供结构性见解， 这种不寻常的二硫化物所表现出的功能不对称性的基础 还原酶。

DESCRIPTION: (provided by applicant) Lactic acid bacteria are known to represent strong paradigms for the biochemical diversity in oxygen metabolism, due to their lack of respiratory cytochromes and other hemeproteins. Instead these organisms rely on networks of unusual flavin-linked respiratory enzymes, and this application is focused on detailed structural and mechanistic studies of three particularly interesting representatives of this group: (1) NADH oxidase (Nox; 2NADH + O2 to 2NAD+ + 2H2O) from the severe human pathogen Streptococcus pyogenes, (2) NADH peroxidase (Npx; NADH + H2O2 - to NAD+ + 2H2O) from Enterococcus faecalis, and (3) alpha-glycerophosphate oxidase (GlpO; alpha-glycerophosphate + O2 - to dihydroxyacetone phosphate + H2O2) from Streptococcus sp. The GlpO sequence is closely related to those of the membrane-associated a-glycerophosphate dehydrogenases (GlpDs); the enzymes are distinguished primarily by the presence of a 50-residue insert unique to GlpO, which has furthermore been shown in limited proteolysis experiments to represent a flexible surface region that plays a crucial role in flavin reduction. Elucidation of the GlpO structure should provide insight into the structures of the related GIpDs as well and should better define the role of the GlpO surface loop during aglycerophosphate oxidation. While the structural homology between Nox and Npx has been reinforced dramatically with new crystal structures during the current project period, only recently has a third member of this small class of NAD(P)H-linked peroxide/disulfide reductases been identified - the FAD-dependent coenzyme A-disulfide reductase (CoADR; NADPH + CoASSCoA to NADP+ + 2CoASH) from the serious human pathogen Staphylococcus aureus. CoADR nicely complements this emerging group of flavoproteins which utilize a single active-site Cys as the basis for an essential redox center - the Cys-sulfenic acid (Cys-SOH) documented with Nox and Npx, and the Cys-SSCoA disulfide present in CoADR. An additional long-term objective of this application is the ongoing definition of the structural, redox, and mechanistic parameters for Cys-SOH function in Nox and Npx, as these enzymes provide the benchmarks by which protein-SOH centers now known to participate in diverse aspects of redox signaling and catalysis are analyzed. The new structures available for Nox and Npx also provide the basis for continuing analyses of the functional distinction between the relatively rare reduction of O2 to 2H2O catalyzed by Nox and the peroxidatic reaction of Npx. The interpretable electron density map recently calculated for CoADR now makes it possible to model and refine the atomic structure of this enzyme as well, thus allowing detailed comparisons with Nox and Npx and providing structural insights into the basis for the functional asymmetry exhibited by this unusual disulfide reductase.