Enzymology of Homoserine Acylation in Methionine Biosynt

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DESCRIPTION (provided by applicant): The methionine biosynthetic pathway, absent in mammals, produces two compounds required for bacterial survival, methionine and S-adenosylmethionine. Disruption of this pathway prevents bacterial growth unless sufficient methionine is obtained from the environment. Therefore, enzymes in this pathway may potentially be targets for novel antibacterial compounds. The first unique step in methionine biosynthesis, acylation of the g-hydroxyl of homoserine, controls flux of homoserine into the pathway. This acylation is catalyzed by one of two enzymes, homoserine transacetylase (HTA) or homoserine transsuccinylase (HTS). The long-term goals of this project are to ascertain whether these enzymes are potential targets for antibacterial agents and to design inhibitors that will function as lead compounds. Initial kinetic characterizations of both enzymes have been reported. In this proposal three specific aims will be pursued. First, the amino acids comprising the Ser-Asp-His catalytic triad of HTA will be identified. This will be accomplished through the combination of sequence alignments, site-directed mutagenesis and steady-state kinetic characterization. Second, the active site residues of HTS, which are different from those of HTA, will be identified. This will be accomplished through a combination of sequence alignment, site-directed mutagenesis, chemical modification, and steady-state kinetic characterization. Third, structural analysis of HTA, HTS, and select mutants will be pursued in an effort to correlate function with structure. All three of these aims directly support the long-term goals of this project. The proposed experiments will be used to train both undergraduate and Master's level students in the area of biochemistry