STRUCTURE AND MECHANISM OF THIOSULFONATE REDUCTASES

硫代磺酸盐还原酶的结构和机制

• 批准号: 7721884
• 负责人: Charles David Stout
• 金额: $ 0.67万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721884
• 关键词: Adenosine; Assimilations; Binding; Complex; Computer Retrieval of Information on Scientific Projects Database; Cysteine; Data; Enzymes; Escherichia coli; Funding; Grant; Inorganic Sulfates; Institution; Mycobacterium tuberculosis; Oxidoreductase; Pathway interactions; Pseudomonas; Reaction; Research; Research Personnel; Resolution; Resources; Role; Source; Structure; Sulfites; Thioredoxin; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; adenylylsulfate reductase; drug development; pathogenic bacteria

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. APS reductase is a key enzyme in the obligate sulfate assimilation pathway of pathogenic bacteria, and is an attractive target for drug development. The enzymes from Mycobacterium tuberculosis and Pseudomonas aeroginosa contain a [4Fe-4S] cluster coordinated by a unique arrangement of cysteines in the primary sequence, CC-x~80-CxxC. A fifth cysteine near the C-terminus is an essential nucleophile that displaces SO32- from the substrate, adenosine-5'-phosphosulfate (APS). The covalent intermediate, Cys249-S-SO3- interacts with the [Fe-S] cluster. We have crystallized this stable reaction intermediate in P. aeroginosa APS reductase, and collected MAD and SAD data at SSRL, to determine the structure at 2.5 ¿ resolution. The subsequent step of the reaction cycle entails reduction of the enzyme bound thiosulfonate by thioredoxin, and release of sulfite. In contrast, the reaction catalyzed by E. coli PAPS (3'-phosphoadenosine-5'-phosphosulfate) reductase, a highly homologous enzyme lacking the cysteine motif, catalyzes the same reaction in the absence of an [Fe-S] cluster. The objective of this project is to define the catalytic mechanism of these two evolutionarily distinct but homologous enzymes, and to identify the role of the [Fe-S] cluster in APS reductase. This requires structure determination of several stable reaction cycle intermediates in APS reductase, and of E. coli PAPS reductase in complex with PAPS.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 APS 还原酶是病原菌专性硫酸盐同化途径中的关键酶，是药物开发的一个有吸引力的靶点。来自结核分枝杆菌和铜绿假单胞菌的酶含有一个 [4Fe-4S] 簇，由一级序列 CC-x~80-CxxC 中半胱氨酸的独特排列协调。 C 末端附近的第五个半胱氨酸是一种重要的亲核试剂，可从底物 5'-磷酸腺苷 (APS) 中置换 SO32-。共价中间体 Cys249-S-SO3- 与 [Fe-S] 簇相互作用。我们在铜绿假单胞菌 APS 还原酶中结晶了这种稳定的反应中间体，并在 SSRL 收集了 MAD 和 SAD 数据，以确定 2.5 ¿ 分辨率的结构。反应循环的后续步骤需要通过硫氧还蛋白还原与酶结合的硫代磺酸盐，并释放亚硫酸盐。相比之下，大肠杆菌 PAPS（3'-磷酸腺苷-5'-磷酸硫酸盐）还原酶（一种缺乏半胱氨酸基序的高度同源酶）催化的反应在不存在 [Fe-S] 簇的情况下催化相同的反应。该项目的目的是确定这两种进化上不同但同源的酶的催化机制，并确定 [Fe-S] 簇在 APS 还原酶中的作用。这需要对 APS 还原酶中的几种稳定反应循环中间体以及与 PAPS 复合的大肠杆菌 PAPS 还原酶进行结构测定。