Mechanisms Involved in Flavin-linked Oxygen Metabolism

黄素相关氧代谢的机制

• 批准号: 6901910
• 负责人: AL CLAIBORNE
• 金额: $ 36.52万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 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 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6901910
• 关键词: Enterococcus; NAD(P)H dehydrogenase; Staphylococcus aureus; Streptococcus pyogenes; active sites; alkaline phosphatase; chimeric proteins; coenzyme analog; crystallization; electron crystallography; enzyme complex; enzyme mechanism; enzyme structure; flavin adenine dinucleotide; flavoproteins; nicotinamide adenine dinucleotide; oxidoreductase; respiratory enzyme; site directed mutagenesis; sulfides

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.

描述：（由申请人提供）已知乳酸菌 代表了氧代谢中生化多样性的强大范式， 由于他们缺乏呼吸性细胞色素和其他止血蛋白。反而 这些生物依赖于异常黄素连接呼吸酶的网络， 该应用集中于详细的结构和机械研究 在该组的三个特别有趣的代表中：（1）纳德 氧化酶（NOX; 2NADH + O2至2NAD + + 2H2O）来自严重的人类病原体 链球菌为链球菌，（2）NADH过氧化物酶（NPX； NADH + H2O2-至NAD + + 2H2O） 从粪肠球菌和（3）α-甘油磷酸氧化酶（Glpo; α-甘油磷酸 + O2-从 链球菌GLPO序列与 膜相关的A-甘油磷酸盐脱氢酶（GLPD）；酶是 主要是由Glpo独有的50分插入物的存在而区别， 此外，在有限的蛋白水解实验中显示了这一点 代表柔性表面区域，在黄素中起着至关重要的作用 减少。阐明GLPO结构应洞悉 相关gipd的结构也应该更好地定义 glpo表面环在氯磷酸磷酸盐氧化过程中。而结构 NOX和NPX之间的同源性已通过新晶体急剧增强 在当前项目期间的结构，直到最近才有第三名成员 在这类小型NAD（P）H-C-C-C-C-C-CHNEC的过氧化物/二硫键还原酶中 已识别 - 依赖FAD的辅酶A-二硫化物还原酶（COADR; NADPH +） 来自严重人类病原体葡萄球菌的Coasscoa至NADP + + 2Coash） 金黄色葡萄酒。 Coadr很好地补充了这一新兴的黄蛋白蛋白 利用单个活动点CYS作为必需氧化还原中心的基础 - 用NOX和NPX记录的Cys-硫酸（CYS-SOH），以及Cys-SSCOA Coadr中存在的二硫键。这是一个额外的长期目标 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中心现在已知参与多样的基准 分析了氧化还原信号传导和催化的各个方面。新结构 可用于NOX和NPX，还为继续分析的基础提供了基础 相对罕见的O2降低至2H2O之间的功能区别 由NOX催化和NPX的过氧化反应。可解释的 现在为COADR计算的电子密度图现在使得 也模型并完善该酶的原子结构，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表现出的功能不对称的基础 还原酶。

项目成果

Crystallization and preliminary crystallographic analysis of the soluble alpha-glycerophosphate oxidase from Streptococcus sp.

链球菌可溶性 α-甘油磷酸氧化酶的结晶和初步晶体学分析。

• DOI: 10.1107/s0907444901018169
• 发表时间: 2002
• 期刊: Acta crystallographica. Section D, Biological crystallography
• 作者: Finnerty,CaseyM;Charrier,Véronique;Claiborne,Al;Karplus,PAndrew
• 通讯作者: Karplus,PAndrew

Studies on the structure and mechanism of Streptococcus faecium L-alpha-glycerophosphate oxidase.

屎链球菌L-α-甘油磷酸氧化酶的结构与机制研究。

• 发表时间: 1986
• 期刊: The Journal of biological chemistry
• 作者: Claiborne,A

Crystal structure and catalytic properties of Bacillus anthracis CoADR-RHD: implications for flavin-linked sulfur trafficking.

炭疽杆菌 CoADR-RHD 的晶体结构和催化特性：对黄素连接的硫运输的影响。

• DOI: 10.1021/bi900887k
• 发表时间: 2009
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Wallen,JamieR;Mallett,TConn;Boles,William;Parsonage,Derek;Furdui,CristinaM;Karplus,PAndrew;Claiborne,Al
• 通讯作者: Claiborne,Al

Analysis of the kinetic and redox properties of NADH peroxidase C42S and C42A mutants lacking the cysteine-sulfenic acid redox center.

缺乏半胱氨酸-次磺酸氧化还原中心的 NADH 过氧化物酶 C42S 和 C42A 突变体的动力学和氧化还原特性分析。

• DOI: 10.1021/bi00002a007
• 发表时间: 1995
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Parsonage,D;Claiborne,A
• 通讯作者: Claiborne,A

Nucleoside triphosphate mimicry: a sugar triazolyl nucleoside as an ATP-competitive inhibitor of B. anthracis pantothenate kinase.

• DOI: 10.1039/b909729e
• 发表时间: 2009-10-07
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: Rowan AS;Nicely NI;Cochrane N;Wlassoff WA;Claiborne A;Hamilton CJ
• 通讯作者: Hamilton CJ