This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Sulfur plays a central role in the metabolism of bacteria. In its reduced form, sulfur is a key component in amino acids, proteins, iron-sulfur clusters and other cofactors such as coenzyme A. Our laboratory has outlined the key features of the sulfate assimilation pathway in the human pathogen, Mycobacterium tuberculosis. In the first committed step in the reduction of sulfate, an iron-sulfur protein termed APS reductase, catalyzes the conversion of the activated form of sulfate, adenosine-5'-phosphosulfate (APS) to sulfite. Notably, APS Reductase is critical for virulence of M. tuberculosis in a murine model of infection. A fundamental question that remains unresolved for this class of enzymes is what role does the iron-sulfur cluster play in catalysis? To investigate this question, and the catalytic mechanism more generally, we have used a combination of kinetics, biophysical analysis and FT-ICR mass spectrometry. Taken together, these data strongly suggest that the iron-sulfur cluster in APS Reductase participates in substrate binding and, likely in its chemical activation, in order to facilitate the catalysis of these biologically important reductions. As this family of enzymes has proved difficult to crystallize, EXAFS represents a complimentary means to directly acquire structural information about the iron-sulfur cluster. To directly test our model, the primary goal of this proposal is to probe the iron-sulfur cluster of M. tuberculosis APS Reductase using EXAFS during discrete stages in the catalytic cycle. Structural data obtained in these studies would be the first detailed structural characterization of an iron-sulfur cluster in sulfonucleotide reductases. Additionally, because APS Reductase represents one of the few validated targets in the effort to develop new leads for drug therapy, molecular details garnered in these studies will be utilized to inform the rational design of small molecules that can be screening for inhibitory activity against APS Reductase.
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