Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations

The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikima te-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcu s pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible c onversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to E PSP and inorganic phosphate. Activation by univalent cations was observed i n the forward reaction, with NH4+, Rb+ and K+ exerting the greatest effects . K-m(PEP) was lowered by increasing [NH4+] and [K+], whereas K-m(S3P) rose with increasing [K+], but fell with increasing [NH4+]. Increasing [NH4+] a nd [K+] resulted in an overall increase in k(cat). Glyphosate (GLP) was fou nd to be a competitive inhibitor with PEP, but the potency of inhibition wa s profoundly affected by [NH4+] and [K+]. For example, increasing [NH4+] an d [K+] reduced K-i(GLP versus PEP) up to 600-fold. In the reverse reaction, the enzyme catalysis was less sensitive to univalent cations. Our analysis included univalent cation concentrations comparable with those found in ba cterial cells. Therefore, the observed effects of these metal ions are more likely to reflect the physiological behavior of EPSP synthase and also add to our understanding of how to inhibit this enzyme in the host organism. A s there is a much evidence to suggest that EPSP synthase is essential for b acterial survival, its discovery in the serious gram-positive pathogen S. p neumoniae and its inhibition by GLP indicate its potential as a broad-spect rum antibacterial target.