Characterization of Homoserine O‐Succinyltransferase: A Key Branch Point Enzyme in an Essential Metabolic Pathway

Homoserine O ‐succinyltransferase (HST) catalyzes the commitment step to methionine production in enteric bacteria, with this pathway branch also leading to other important biological precursors in bacteria, plants and fungi. This enzyme is not found in humans, and the essential nature of this enzymatic pathway in microorganisms makes HST an attractive new target for antimicrobial design. The HST from Escherichia coli has been cloned, purified and kinetically characterized. Through site‐directed mutagenesis and steady‐state kinetics the residues that comprise a catalytic triad for HST, the catalytic cysteine nucleophile, an active site acid‐base histidine, and the base orienting glutamate, have been identified and characterized. Several residues which confer substrate specificity for both homoserine and succinyl‐CoA have also been identified and characterized. Mutations of an active site glutamate to either an aspartic acid or alanine have been shown to drastically alter the K m for homoserine, allowing assignment of this glutamate as interacting with the α‐amino group of homoserine. An active site arginine helps to orient the carboxyl moiety of homoserine, while the carboxyl moiety of succinyl‐CoA is positioned for catalysis by a lysyl residue. Removing functionality at either of these positions by mutagenesis alters the enzyme's ability to effectively utilize homoserine or succinyl‐CoA, respectively, reflected in an increased K m and decreased catalytic efficiency. The information obtained from this active site mapping is being used to identify selective inhibitors of this critical branch point enzyme that can serve as lead compounds for antimicrobial development.