title: Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line.
abstract: Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 mM glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogeneous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with Mr 49,000-55,800. Km values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 18 microM, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate (Ki = 0.17 microM). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology*; D007118:Immunoassay; D007700:Kinetics; D063245:Plant Development; D010944:Plants / Q000201:enzymology*; D014166:Transferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D005998:Glycine; D006540:Herbicides; D014166:Transferases; D000097797:Glyphosate