Bacterial survival strongly depends on the functionality of a unique class of enzymes, the enolpyruvyl transferases. The only representatives of this family are MurA and EPSP synthase. MurA catalyzes the first committed step in the biosynthesis of the bacterial cell wall; EPSP synthase is the sixth enzyme of the shikimate pathway leading to the synthesis of aromatic compounds in numerous microorganisms. Since both pathways are absent from mammals, enolpyruvyl transferases are attractive targets for antibiotic drugs. The reactions catalyzed by enolpyruvyl transferases proceed through the chemically unusual transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to a second substrate. The uniqueness of these enzymes is also reflected by their distinct 3-dimensional architecture: two globular, inside-out, alpha-beta-barrel domains are connected by a double-stranded hinge. A number of recent structural studies revealed that the two domains approach each other on substrate binding, such that the active site emerges in the interdomain cleft. The central goal of this proposal is to understand how the unusual reaction mechanism, the distinct structural properties, and the dynamics of enolpyruvyl transferases are related to each other.
The specific aims are: 1) to visualize the principle steps of enolpyruvyl transfer by determining the high resolution X-ray structures of binary and ternary enzyme:substrate complexes. Single-site mutant enzymes with altered kinetics and dynamics shall be selected to trap the reaction at different states; 2) to characterize the structural prerequisites and the principles of the induced-fit mechanism by fluorescence studies and X-ray crystallographic analysis of mutant enzymes from MurA and EPSP synthase; 3) to analyze the structure and function of enolpyruvyl transferases from pathogenic bacteria in order to reveal how the requirements for enolpyruvyl transfer are realized in enzymes with low sequence homologies to E. coli and 4) to employ the aptamer technology in order to create novel inhibitors for MurA and EPSP synthase. These studies will contribute directly to the basic knowledge of protein structure families, structural folds, and the relationship between structure and function in enzymatic reactions. Furthermore, the proposed work will provide deep insight in the potential of enolpyruvyl transferases as broad-spectrum antibacterial targets and shall facilitate the design of novel antibiotic drugs.
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