Mechanisms of Enzyme Catalysis

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DESCRIPTION (provided by applicant): The goal of this project is to determine structure, function, and mechanism of action of selected members of the PEP mutase/isocitrate lyase and 4-hydroxybenzoyl-CoA thioesterase enzyme families. This information will be used to relate active site structure to catalysis, and thereby identify markers, which can be applied in the assignment of function to all unknown proteins within each enzyme family. The novel protein functions and metabolic pathways that are anticipated to emerge from these efforts will, along with the active site structure determinations, serve as the foundation for drug discovery. Finally, from the proposed studies the principal investigator and her group will gain insight into the catalytic mechanisms of the enzymes mediating the diverse chemistries represented by the two enzyme families, and into how these catalytic mechanisms evolved from ancestral active site templates. Specific Aims 1-4 will address structure, function and catalytic mechanism in four members of the PEP mutase/ isocitrate lyase enzyme family: phosphonopyruvate hydrolase of Burkholderia cepacia, 5, 1 0-methylenetetrahydrofolate: 3-methyl-2-oxobutanoate hydroxymethyl transferase of Pseudomonas aeruginosa, a protein associated with petal death in carnation, and a protein of unknown function present in Mycobacterium tuberculosis (Rv1998c). Specific Aims 5-9 will address structure, function and catalytic mechanism in five members of the 4-hydroxybenzoyl-CoA thioesterase enzyme family: 4-hydroxybenzoyl-CoA thioesterase, the YgbC enzyme of the Haemophilus influenza Tol-pal Pathway, the P76084 unknown protein of the E.coli Phenylacetate Catabolic Pathway, the BH1 997 unknown protein of the Bacillus halodurans Upper Gentisate Pathway and the Pseudomonas aeruginosa PA551 9 homologue to the Human Long Chain Acyl-CoA Thioesterase