title: Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations.
abstract: The aroA gene (Escherichia coli nomenclature) encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the gram-positive pathogen Streptococcus pneumoniae has been identified, cloned and overexpressed in E. coli, and the enzyme purified to homogeneity. It was shown to catalyze a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate. Activation by univalent cations was observed in the forward reaction, with NH+4, Rb+ and K+ exerting the greatest effects. Km(PEP) was lowered by increasing [NH+4] and [K+], whereas Km(S3P) rose with increasing [K+], but fell with increasing [NH+4]. Increasing [NH+4] and [K+] resulted in an overall increase in kcat. Glyphosate (GLP) was found to be a competitive inhibitor with PEP, but the potency of inhibition was profoundly affected by [NH+4] and [K+]. For example, increasing [NH+4] and [K+] reduced Ki(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 bacterial 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. As there is a much evidence to suggest that EPSP synthase is essential for bacterial survival, its discovery in the serious gram-positive pathogen S. pneumoniae and its inhibition by GLP indicate its potential as a broad-spectrum antibacterial target.
authors.affiliation: Anti-Infectives Research, SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426, USA.
authors.forename: W
authors.initials: W
authors.lastname: Du
authors.forename: N G
authors.initials: NG
authors.lastname: Wallis
authors.forename: M J
authors.initials: MJ
authors.lastname: Mazzulla
authors.forename: A F
authors.initials: AF
authors.lastname: Chalker
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: W S
authors.initials: WS
authors.lastname: Liu
authors.forename: H
authors.initials: H
authors.lastname: Kallender
authors.forename: D J
authors.initials: DJ
authors.lastname: Payne
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis / Q000187:drug effects; D002412:Cations / Q000494:pharmacology*; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010728:Phosphoenolpyruvate / Q000378:metabolism / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D002412:Cations; D005998:Glycine; D007770:L-Lactate Dehydrogenase; D010728:Phosphoenolpyruvate; D011188:Potassium; D011770:Pyruvate Kinase; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D012765:Shikimic Acid; D000097797:Glyphosate; C042786:shikimic acid-3-phosphate