Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L.

Mousdale, David M.; Coggins, John R.

doi:10.1007/bf00392469

Cited by 87 publications

(33 citation statements)

References 26 publications

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“…In N. silvestris, uncompetitive inhibition by Pi with respect to shikimate-3-P and mixed inhibition by Pi with respect to PEP were noted utilizing a partially purified EPSP synthase preparation in experiments where the fixed substrate was present at saturating concentration. These product inhibition patterns imply that the N. silvestris enzyme possesses an order of substrate binding that would be consistent with the mechanism proposed by Boocock and Coggins (6) (27) and mung bean (21), experiments are in progress to determine whether two spatially separate enzyme forms might be present in N. silvestris. Column chromatography has revealed two closely overlapping peaks of EPSP synthase activity that eluted from DEAE-cellulose.…”

Section: Discussionsupporting

confidence: 80%

“…In both cases, intracellular accumulation of shikimate-3-P is not expected to overcome glyphosate inhibition, as would be expected in the case ofstrictly competitive inhibition. Consistent with all systems so far examined (3,6,27), glyphosate inhibition of N. silvestris EPSP synthase was strictly competitive with respect to PEP. That glyphosate is an exceedingly potent enzyme inhibitor is reflected by the following K, (PEP) values reported to date: Pisum sativum, 0.08 uM (27); K. pneumoniae, I gM (3); N. crassa, 1.1 Mm (6) Mechanistic studies, utilizing partially purified bacterial enzyme (5,15), suggest that EPSP synthase proceeds via a reversible addition-elimination mechanism in which the enolpyruvyl moiety of PEP is transferred apparently unchanged to shikimate-3-P.…”

Section: Discussionsupporting

confidence: 79%

“…Consistent with all systems so far examined (3,6,27), glyphosate inhibition of N. silvestris EPSP synthase was strictly competitive with respect to PEP. That glyphosate is an exceedingly potent enzyme inhibitor is reflected by the following K, (PEP) values reported to date: Pisum sativum, 0.08 uM (27); K. pneumoniae, I gM (3); N. crassa, 1.1 Mm (6) Mechanistic studies, utilizing partially purified bacterial enzyme (5,15), suggest that EPSP synthase proceeds via a reversible addition-elimination mechanism in which the enolpyruvyl moiety of PEP is transferred apparently unchanged to shikimate-3-P. It has been proposed that EPSP synthase from N. crassa (6) and Escherichia coli (24) sequentially binds shikimate-3-P and PEP as the first and second substrates, respectively.…”

Section: Discussionsupporting

confidence: 79%

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Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Rubin

Gaines²,

Jensen³

1984

Plant Physiol.

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Treatment of isogenic suspension-cultured cells of Nicotiana silvestrisSpeg. et Comes with glyphosate (N-phosphonomethyljglycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-D-arabiaoheptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ionexchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK. values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO-CH2NH2+CH2PO32-, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K,' = 183 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an lenzyme:shikimate-3-PJ complex and ultimately forms the dead-end complex of lenzyme:shikimate-3-P:glyphosatel.Glyphosate (N-[phosphonomethyl]glycine) is a broad-spectrum herbicide which is also a potent growth inhibitor ofbacteria and algae (14). Although glyphosate may ultimately perturb a variety of biochemical processes including protein synthesis, nucleic acid synthesis, photosynthesis and respiration (16), the early conjecture (18)

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Section: Discussionsupporting

confidence: 80%

Section: Discussionsupporting

confidence: 79%

Section: Discussionsupporting

confidence: 79%

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Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Rubin

Gaines²,

Jensen³

1984

Plant Physiol.

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“…Previously [43], an M, of 35000 had been determined for the Aerobacter aerogenes (= K. pneumoniae) enzyme by sucrose density gradient centrifugation. The subunit M, of EPSP synthases from E. coli and pea seedlings were reported to be 49000 and 50000 respectively, while the native M , values were 42000 and 44000, respectively [20,21]. Quite recently nucleotide sequence analysis of the cloned EPSP synthase gene from Salmonella typhimurium has been carried out and a putative reading frame for a polypeptide with a M , of 46000 was identified [44].…”

Section: Discussionmentioning

confidence: 99%

5‐Enolpyruvylshikimate‐3‐phosphate synthase of Klebsiella pneumoniae

Steinrücken

Amrhein

1984

European Journal of Biochemistry

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The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1 -carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62-1 (= Klebsiellapneumoniae ATCC 25306). A 3300-fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE-cellulose, Sephadex G-75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49 %.An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of M, = 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme-catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3-phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 6 0 T , the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations.Non-linear Lineweaver-Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme-substrate complexes : forward reaction: 43 pM (PEP) and 22 pM (S3P); reverse reaction: 1.3 pM (EPSP) and 2.6 mM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction.The shikimate pathway represents the common biogenetic background for a large number of pre-aromatic and aromatic natural compounds, in particular the aromatic amino acids, in microorganisms and plants [1,2]. In the course of the biosynthetic transformations of the pre-aromatic intermediates, an intact enolpyruvyl (carboxyvinyl) group is transferred from phosphoenolpyruvate (PEP) to the 5-hydroxyl group of shikimate 3-phosphate to give rise to 5-enolpyruvylshikimate 3-phosphate (EPSP) and inorganic phosphate [3]. This transfer, which is catalyzed by EPSP synthase (3-phosphoshikimate-lcarboxyvinyl transferase) represents a rare type of reaction, which has a parallel only in the reaction catalyzed by enoylpyruvate transferase (phosphoeno1pyruvate:UDP-2-acetamido-2-deoxy-~-g~ucose 2-enoyl-1-carboxyethyltransferase), an enzyme involved in the biosynthesis of the bacterial cell wall peptidoglycan [4,5]. For EPSP synthase, Levin and Sprinson [3] proposed a reversible addition/elimination mechanism, which was supported by later isotope-exchange studies [6,7], but these experiments were carried out with only partially purified bacterial extracts. We have recently shown that glyphosate [N-(phosphonomethy1)-glycine], a highly efficient and widely used broad-spectrum herbicide 181, is a Part of the Doctoral Dissertation of H...

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“…In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M,) 53900 ± 900 as compared to M, 45500 ± 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures.The nonselective, broad spectrum herbicide glyphosate, N-(phosphonomethyl)glycine, has been shown to be a potent and efficient inhibitor of the shikimate pathway enzyme EPSPsynthase2 from plants and microorganisms (1,4,11,22 (Sigma) were used for precipitation with either preimmune serum (5 Ml) or the antiserum (20 Ml). The latter had been affinity-purified using EPSP-synthase bound to nitrocellulose (A. Schulz, personal communication).…”

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confidence: 99%

5-Enolpyruvylshikimate 3-Phosphate Synthase, the Target Enzyme of the Herbicide Glyphosate, Is Synthesized as a Precursor in a Higher Plant

Holländer-Czytko

Amrhein²

1987

Plant Physiol.

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Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30-to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M,) 53900 ± 900 as compared to M, 45500 ± 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures.The nonselective, broad spectrum herbicide glyphosate, N-(phosphonomethyl)glycine, has been shown to be a potent and efficient inhibitor of the shikimate pathway enzyme EPSPsynthase2 from plants and microorganisms (1,4,11,22 (Sigma) were used for precipitation with either preimmune serum (5 Ml) or the antiserum (20 Ml). The latter had been affinity-purified using EPSP-synthase bound to nitrocellulose (A. Schulz, personal communication). Radioactive proteins were eluted from the protein A complex and analyzed by SDS-gel electrophoresis on a 8 to 13% polyacrylamide gel (9). The gel was incubated with Enhance (NEN), dried, and exposed to a Kodak XAR5 film. Fluorograms were photographed, printed on an Agfaortho 25 document copying film, and scanned at 620 nm with an Isco gel scanner for quantification. For analysis of immunoprecipitable material by SDS-gel electrophoresis, between 2 and 4 translation assays were combined. About 0.1% of radioactivity incorporated into protein could be recovered in the immunoprecipitates from assays containing RNA from nonadapted cells as compared to 0.8 to 1% from adapted cells (Table I). Further analysis by SDS-electrophoresis and fluorography revealed that the immunoprecipitable material was concentrated in a single band of Mr 53900 ± 900 (mean ± SD of 7 independent experiments) ( Fig. 1). As precipitation with preimmune serum produced no radioactive band (data not shown) and as affinity-purified anti4EPSP-synthase) serum was employed, we conclude that the band represents a higher mol wt precursor of EPSP-synthase, the mature form of which has a Mr of 45,500 ± 1,000 (20

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Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from seedlings of Pisum sativum L.

Cited by 87 publications

References 26 publications

Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

5‐Enolpyruvylshikimate‐3‐phosphate synthase of Klebsiella pneumoniae

5-Enolpyruvylshikimate 3-Phosphate Synthase, the Target Enzyme of the Herbicide Glyphosate, Is Synthesized as a Precursor in a Higher Plant

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