STRUCTURE AND MECHANISM OF THIOSULFONATE REDUCTASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 APS还原酶是病原菌专性硫酸盐同化途径中的关键酶，是药物开发的重要靶点。结核分枝杆菌和铜绿假单胞菌的酶含有一个[4Fe-4S]簇，其一级序列CC-x~80-CxxC由半胱氨酸的独特排列所协调。C末端附近的第五个半胱氨酸是一种重要的亲核试剂，可以取代底物硫代磷酸腺苷(APS)中的SO32-。共价中间体Cys249-S-SO3-与[Fe-S]团簇相互作用。我们在铜绿假单胞菌APS还原酶中结晶了这个稳定的反应中间体，并在SSRL收集了MAD和SAD数据，以2.5？分辨率确定了结构。反应循环的后续步骤需要硫氧还蛋白还原酶结合的硫代磺酸盐，并释放亚硫酸盐。相反，在缺乏半胱氨酸基序的高度同源酶--大肠杆菌PAPPS(3‘-磷酸腺苷-5’-磷酸硫酸盐)还原酶的催化下，在没有[Fe-S]簇的情况下，该反应也能催化同样的反应。本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。这需要确定APS还原酶中几个稳定的反应循环中间体的结构，以及大肠杆菌PAPS还原酶与PAPS的复合体的结构。

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.