title: Determination of enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase by LC/MS.
abstract: A liquid chromatography-mass spectrometry (LC/MS) method for determining the enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), an enzyme of the shikimate pathway, was developed. EPSP synthase catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S-3-P) and phosphoenolpyruvate (PEP) in microorganisms and plants. The enzymatic activity of EPSP synthase was assessed by the determination of EPSP after a 30-min incubation with S-3-P and PEP using the LC/MS system. EPSP synthase activity is given in terms of the produced EPSP (pmol/min/mg protein). Glyphosate (N-phosphonomethyl glycine)-tolerant EPSP synthase from the Agrobacterium sp. strain CP4 (CP4-EPSP synthase) in genetically modified soybeans (GM-soybeans) was found to have an enzymatic activity of 736 EPSP pmol/min/mg protein in the presence of 3 nmol of S-3-P. In contrast, the enzyme activity of non-GM-soybeans was 21 EPSP pmol/min/mg protein. The EPSP synthase activity was markedly decreased in the non-GM-soybeans by the addition of glyphosate, but the enzyme activity of the GM-soybeans was only slightly decreased with this treatment. This LC/MS system could also be applicable to the measurement of EPSP synthase activity in different plant species and the detection of herbicide-tolerant EPSP synthase in GM foods.
authors.affiliation: National Institute of Health Sciences: 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Haruyo
authors.initials: H
authors.lastname: Okunuki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Akiyama
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.forename: Akihiro
authors.initials: A
authors.lastname: Hino
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Goda
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada
authors.forename: Masatake
authors.initials: M
authors.lastname: Toyoda
authors.forename: Tamio
authors.initials: T
authors.lastname: Maitani
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology; D012765:Shikimic Acid / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology
chemical_list: D012765:Shikimic Acid; D010728:Phosphoenolpyruvate; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase