STRUCTURE AND MECHANISM OF THIOSULFONATE REDUCTASES

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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.