This revised renewal proposal outlines four specific objectives for work in the shikimate pathway aimed at understanding key enzymatic processes and regulatory phenomena that will have important biomedical applications.
Specific aims of the project include: Studies on Chorismate Mutases: The importance of prephenate binding as a target for inhibitor design will be probed by studying the x-ray crystal structures of Escherichia coli mutases bound to prephenate and to the selective mutase inhibitor S- (-)-dinitrobiphenic acid (S-DNBA). Such studies may elucidate inhibitor selectivity and resolve questions about the catalytic mechanism. Computational ligand-docking and molecular modeling approaches will be used to develop more potent ligands for the chorismate and prephenate-binding pockets, respectively. Studies on Prephenate-Aromatizing Enzymes: Crystallography and solution NMR will be used to elucidate the active site structures of prephenate dehydratase (PDT) and catalytically inactive T278A mutants in the presence of S-DNBA and prephenate, respectively. The resulting structural information together with site-directed mutagenesis will be used to develop a catalytic mechanism for these enzymes. Domain mapping and complementation studies on the bifunctional T-protein are proposed to elucidate the spatial arrangement of CM and prephenate dehydrogenase (PDH) sites. A monofunctional PDH will be subcloned to elucidate its catalytic mechanism. Studies on Bifunctional Proteins in Phenylalanine and Tyrosine Biosynthesis: Engineered proteins co-crystallized with S-DNBA and/or prephenate bound in the mutase and dehydratase active sites will be used to study how substrate is channeled from CM to PDT in the Phe biosynthesis. Cooperative effects will be measured using microcalorimetry. Modeling studies are proposed to detect the role of an electrostatic gradient in channeling. A similar approach is proposed to probe spatial relationships of the CM/PDH sites in the T-protein. Such studies may reveal non-obvious strategies for designing potent enzyme inhibitors. Studies on Feedback Inhibition at the Chorismate Branchpoint: X-ray and NMR analysis of the P-protein's R domain complexed with Phe should reveal structural changes involves in feedback inhibition. A similar approach, involving mutagenesis of conserved residues and structural studies, is proposed to elucidate the T-protein's R-domain. R-Domain swapping experiments will establish whether feedback control elements in the P/T proteins are interchangeable, and whether different mechanisms are involved.
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