TARGETING 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE AND THE SHIKIMATE PATHWAY

Bacteria, plants, fungi, and apicomplexan parasites require the functionality of the shikimate pathway for biosynthesis of essential aromatic compounds. Animals lack the enzymes that constitute the shikimate pathway, making these attractive antimicrobial targets. Glyphosate (the active ingredient in Roundup herbicide) inhibits the shikimate pathway enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), but has limited antimicrobial activity. Several EPSPS point-substitutions can induce glyphosate tolerance, and some species possess EPSPS with intrinsic glyphosate insensitivity. To aid development of second-generation EPSPS inhibitors, I probed the inhibition of glyphosate-tolerant EPSPS. I examined the effects of mutations at Pro101 and Thr97 on the structure, function, and glyphosate sensitivity of E. coli EPSPS; I kinetically characterized the glyphosate-tolerant Staphylococcus aureus EPSPS and Agrobacterium sp. strain CP4 EPSPS and determined the three-dimensional structure of the CP4 enzyme; I studied the interaction of EPSPS with analogs of the substrate and the tetrahedral intermediate; and I conducted high throughput screening to identify novel EPSPS inhibitors