Coenzyme A (CoA) is the major acyl group carrier for all organisms and is essential for cell survival. In bacteria, the last two reactions of CoA biosynthesis are mediated by the enzymes phosphopantetheine adenylyltransferase (PPAT), which generates dephospho-CoA (dPCoA), and dephospho-CoA kinase (DPCK), which yields CoA. In contrast, in higher eukaryotes a bifunctional enzyme, CoA synthase, catalyzes both of these final reactions. The substrate of PPAT is rate-limiting and the enzyme is feedback regulated by CoA, indicating that PPAT is an excellent target for developing novel antibiotics. We have solved the crystal structures of PPAT from Escherichia coli, which exists as a hexamer, and of PPAT bound to its substrates and to its product dPCoA. These studies identified the residues involved in substrate/product binding and revealed an in-line displacement catalytic mechanism. Finally, our Preliminary Studies of the PPAT:CoA crystal structure revealed an unusual mechanism of feedback inhibition of PPAT by CoA. The structure of the monomeric DPCK enzyme (from Haemophilus influenzae) has recently been solved and suggests an induced-fit reaction mechanism typical of kinases. Collectively, these structures form the foundation for the design of PPAT- and DPCK-specific inhibitors, an important issue in the context of treating drug-resistant bacteria, in particular tuberculosis. Like other bacteria, Mycobacterium tuberculosis requires PPAT and DPCK, and although the catalytic mechanisms of tuberculin PPAT and DPCK are likely identical to their homologs in other bacteria, there are significant differences predicted for the structures of these enzymes, especially DPCK. Recombinant M. tuberculosis PPAT and DPCK proteins have been produced and we have crystallized tuberculin PPAT. Experiments in Specific Aims #1 and #2 will determine the native and substrate-bound crystal structures of M. tuberculosis PPAT and DPCK. In addition, the structures of tuberculin PPAT bound to its product dPCoA, and to its inhibitor CoA will be solved. The design of PPAT- and DPCK-specific inhibitors requires that they not inhibit human CoA synthase. The monomeric nature, predicted structure, and coupled catalytic functions of CoA synthase indicate that regulation of this enzyme is unique, and a novel N-terminal domain may play a regulatory role. Experiments in Aim #3 will determine the native and substrate-bound crystal structures of human CoA synthase. The proposed crystallographic studies are essential for the development of novel inhibitors that selectively target M. tuberculosis PPAT and DPCK.
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