The objective of this work is to investigate the structure, mechanism of action, and inhibition of three different types of metalloprotein, all of which utilize [4Fe-4S] clusters in catalysis. The reactions catalyzed are reductive dehydroxylations, dehydrations and isomerizations, and all of the proteins are of long-term interest because they are essential for the survival of many pathogens and are not used by humans. The proteins are IspG and IspH, involved in reductive dehydroxylations in isoprenoid biosynthesis;quinolinate synthase (NadA), involved in NAD biosynthesis;and dihydroxyacid dehydratase (DHAD) and isopropylmalate isomerase (LeuCD), involved in amino-acid biosynthesis.
In Aim 1 the first objective is to investigate a previously uncharacterized class of IspHs that are found in many anaerobic bacteria, such as those which cause tetanus and botulism, as well as many which are found in the human microbiome. These proteins are 2-3x larger than most bacterial IspHs and contain an IspH-RPS1 (ribosomal binding protein S1) fusion and might act as iron or oxygen sensors. The second objective is to probe the mechanisms of action of both IspG and IspH by using rapid freeze-quench EPR, calorimetry, site-directed mutagenesis, M?ssbauer, X-ray crystallography, and DFT to characterize reaction intermediates. The third objective is to determine the structures of the 3-domain IspGs and develop novel IspG inhibitors.
Aim 2 involves investigation of NadA, an enzyme whose apo-structure resembles that of IspH but whose structure with its 4Fe-4S cluster is unknown. The objective is to determine its structure and mechanism of action and to develop inhibitors as drug leads (against the organism that causes gastric ulcers and carcinoma).
The third Aim i nvolves two [4Fe-4S] hydratase/isomerases (DHAD and LeuCD). Both are predicted to contain two domains and it appears that there are structural similarities between DHAD, LeuCD, aconitase and fumarase A, a hypothesis we will test. Inhibitors have been reported as herbicides, blocking leucine and other branched chain amino-acid biosynthesis, an effect that with the LeuCD inhibitor is reversed with L- leucine, and M. tuberculosis leuCD knockouts are being developed as TB vaccines, making LeuCD and DHAD potential new drug targets and here, we will develop inhibitors, active in cells, that bind to their [4Fe-4S] clusters.
The project is aimed at understanding the structure and function of a series of metallo-proteins that contain [4Fe-4S] clusters. These proteins are essential for the growth and survival of the organisms that cause tuberculosis, stomach ulcers and malaria but are not used by humans, making them of particular interest as drug targets.
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