Properties of the 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms isolated from maize cultured cells (original) (raw)
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Functional Plant Biology, 2002
The expression of two 5-enol-pyruvyl-shikimate-3-phosphate synthase (EC 2.5.1.19) isoforms was investigated in Zea mays L. suspension-cultured cells following exposure to a fungal elicitor. Activity levels of isozyme II specifically increased soon after treatment, in strict connection with induction of phenylalanine ammonia-lyase (PAL) and attainment of a new free-phenylalanine homeostasis at a higher concentration. However, a few days later, activity of the other enzyme form was also significantly enhanced, concomitant with a sharp rise in overall amino acid content, a further increase in PAL level and a resumption of cell lysis. Besides strengthening the hypothesis that an entire set of genes encoding for shikimate pathway enzymes (whose expression is specifically involved in plant dynamic defence) may exist, a general change in the levels of several amino acids seems to point towards a reprogramming of their metabolism in elicited cells.
Plant Physiology, 1994
l h e shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3phosphate (EPSP) synthase (3-phosphoshikimate-1-carboxyvinyl transferase, EC 2.5.1.19) was purified from cultured maize (Zea mays 1. var Black Mexican Sweet) cells. Homogeneous enzyme preparations were obtained by a four-step procedure using ammonium sulfate fractionation, anion-and cation-exchange chromatography, and substrate elution from a cellulose phosphate column. l h e last step resulted in two well-separated adivities of about the same molecular weight. A 2000-to 3000-fold purification, with an overall recovery of one-fourth of the initial adivity, was achieved. Both EPSP synthase isoforms were characterized with respect to structural, kinetic, and biochemical properties. Only slight differences are seen in molecular mass, adivation energy, and apparent affinities for the two substrates. A more pronounced difference was found between their thermal inactivation rates. l w o EPSP synthase isoforms were also elucidated in crude homogenates by anion-exchange fast protein liquid chromatography. lhis allowed us to follow their expression during a culture growth cycle. One form was found at substantial levels throughout, whereas the other increased in exponentially growing cells and declined in latelogarithmic phase. l h e analysis of highly purified plastid preparations demonstratèd a plastidial localization of both proteins. Possible functional roles for maize EPSP synthase isozymes, with regard to the dual-pathway hypothesis and to the recent findings on defense-related aromatic biosynthesis in higher plants, are discussed.
Enzymological Basis for Herbicidal Action of Glyphosate
Plant Physiology, 1982
The effects of 1 milliolar glyphosate (N-Iphosphonomethyllglycine) upon the activities ofenzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata IL.I Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-Darabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by MnI2+ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co2, for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co02 is a V33.. activator with respect to both substrates, glyphosate cannot act simply by Co2' chelation because inhibition is competitive with respect to erythrose4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimc acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal ofgrowth inhibition in 7-day seedlfings ofmung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target. Glyphosate (N-[phosphonomethyl]glycine) is an exceedingly effective broad-spectrum herbicide which is readily translocated between plant tissues (29). Residual glyphosate also biodegrades very satisfactorily in soil environments (32). Beyond its herbicidal properties, glyphosate is often a potent inhibitor of microbial growth. The general finding that glyphosate inhibition is antagonized by one or more of the aromatic amino acids extends to a wide range of organisms, including prokaryotes, eukaryotic algae, and plants (15). In buckwheat and cultured cells of Galium mollugo L. (Rubiaceae), physiological experiments have shown that glyphosate causes shikimate accumulation and blocks the entry of radiolabeled shikimate into aromatic amino acids (1). Evidently, the accumulation of shikimate in response to glyphosate treatment generally occurs in plants, inasmuch as such data have now been obtained from dozens of diverse species (2, 3). These data are consistent with the possibility that EPSP2 synthase might be the 'Supported by Department of Energy Contract EP-78-024967
Journal of Chromatography A, 1986
The plant and microbial enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (E .C. 2.5 .1 .19; alternative name 3-phosphoshikimate lcarboxyvinyltransferase) is inhibited by the broad-spectrum herbicide glyphosate (N-phosphonomethylglycine) and is the principal site of the biological action of this xenobiotic'-e. EPSP synthase is present in plant tissues in relatively small quantities (0.03-0 .06% of the total soluble protein 7' 9) but has been purified to homogeneity from pea seedlings 9 and a plant cell culture'. While the synthesis and cellular levels of the enzyme can be selectively increased in cell cultures by adaptation to the presence of glyphosate'', the purification of EPSP synthase from plant tissues is in general a lengthy process and yields only microgram quantities of the pure enzyme. EPSP synthase is predominantly a chloroplastic enzyme in pea seedling tissues °a nd is present in chloroplast lysates at higher specific activities than in crude tissue extracts 9,10. We have utilized this compartmentation of the enzyme to develop a rapid small-scale purification of EPSP synthase from a range of higher plant species. EXPERIMENTAL Plant materials Pea (Pirum sativum L. c .v. "Onward") seeds were germinated and grown in growth chambers as described previously 9. Maize (Zea mays L.) plants were grown in similar growth chambers ; leaf tissue was used from 10-day-old plants. Spinach (Spinacia oleracea L.) plants were greenhouse-grown ; leaves were taken from approximately 6-week-old plants. Lettuce (Lactuca saliva L.) was obtained commercially. Chloroplast preparation Washed chloroplast preparations were made from 100-500 g fresh weight of leaf or shoot tissue by the method of Nakatani and Barber". Fast protein liquid chromatography Washed chloroplasts were lysed by dilution in 10-40 ml of 20 mM Tris (pH 7.5 with hydrochloric acid) containing 0 .1 mM dithiothreitol (DTT) and 1 mM benzamidine hydrochloride (buffer A). The lysate was centrifuged at 80 000 g for I h at
The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids in Arabidopsis thaliana
The Arabidopsis Book, 2010
The aromatic amino acids phenylalanine, tyrosine and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway, with chorismate serving as a major branch point intermediate metabolite. Yet, the regulation of their synthesis is still far from being understood. So far, only three enzymes in this pathway, namely, chorismate mutase of phenylalanine and tyrosine synthesis, tryptophan synthase of tryptophan biosynthesis and arogenate dehydratase of phenylalanine biosynthesis, proved experimentally to be allosterically regulated. The major biosynthesis route of phenylalanine in plants occurs via arogenate. Yet, recent studies suggest that an alternative route of phynylalanine biosynthesis via phenylpyruvate may also exist in plants, similarly to many microorganisms. Several transcription factors regulating the expression of genes encoding enzymes of both the shikimate pathway and aromatic amino acid metabolism have also been recently identifi ed in Arabidopsis and other plant species. Figure 2. The shikimate pathway. Enzymes involved in the biosynthesis of chorismate.
5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, an enzyme used in biosynthesis of aromatic amino acids in plants, many bacteria, and microbes, is a prime target for drugs and herbicides. The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity as it is competitive with respect to phosphoenolpyruvate and uncompetitive with respect to shikimate-3-phosphate. It is important to study this enzyme for elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.
Regulatory Toxicology and Pharmacology, 2009
Glyphosate tolerance can be conferred by decreasing the herbicide's ability to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase, which is essential for the biosynthesis of aromatic amino acids in all plants, fungi, and bacteria. Glyphosate tolerance is based upon the expression of the double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) protein. The 2mEPSPS protein, with a lower binding affinity for glyphosate, is highly resistant to the inhibition by glyphosate and thus allows sufficient enzyme activity for the plants to grow in the presence of herbicides that contain glyphosate. Based on both a review of published literature and experimental studies, the potential safety concerns related to the transgenic 2mEPSPS protein were assessed. The safety evaluation supports that the expressed protein is innocuous. The 2mEPSPS enzyme does not possess any of the properties associated with known toxins or allergens, including a lack of amino acid sequence similarity to known toxins and allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenous or oral administration (at 10 or 2000 mg/kg body weight, respectively). In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the 2mEPSPS protein in human food or in animal feed.
Proceedings of the National Academy of Sciences, 1986
5- enol Pyruvylshikimate-3-phosphate synthase (EPS P synthase; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of the shikimate pathway in plants. This enzyme is the target for the nonselective herbicide glyphosate ( N -phosphonomethylglycine). We have previously isolated a full-length cDNA clone of EPS P synthase from Petunia hybrida . DNA sequence analysis suggested that the enzyme is synthesized as a cytosolic precursor (pre-EPS P synthase) with an amino-terminal transit peptide. Based on the known amino terminus of the mature enzyme, and the 5′ open reading frame of the cDNA, the transit peptide of pre-EPS P synthase would be maximally 72 amino acids long. To confirm this prediction and to assay directly for translocation of pre-EPS P synthase into chloroplasts in vitro , we cloned the full-length cDNA into an SP6 transcription system to produce large amounts of mRNA for in vitro translation. The translation products, when analyzed b...
Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice
Plant Biotechnology, 2009
Tryptophan (Trp) and phenylalanine (Phe) are essential aromatic amino acids that animals cannot synthesize and are dependent on plants for their supply. In particular, Trp contributes to the nutritional quality of plant-based foods, and, along with lysine, methionine and threonine, it is used to supplement animal feeds. The other aromatic amino acids, Phe and tyrosine (Tyr), are used for the production of the low-calorie sweetener aspartame and the anti-Parkinson's disease drug L-dopa, respectively. The biosynthetic pathways for aromatic amino acids and their regulation have been extensively explored in bacteria because of their utility in the food and drug industry. In plants, aromatic amino acids are the precursors for a large variety of secondary metabolites including indole alkaloids, phenylpropanoids, flavonoids, and the phenolic polymer, lignin. One of plant growth regulators, indole-3-acetic acid (IAA), has also been shown to be biosynthesized from Trp. Thus, aromatic amino acids also play important roles in plant defense and development. In bacteria, fungi and plants, the three aromatic amino acids, Trp, Phe and Tyr, are synthesized from a common precursor, chorismate that originates from the shikimate pathway (Figure 1). In bacteria, this pathway is almost exclusively used to produce aromatic amino acids for protein synthesis but, in higher plants, this pathway leads to the production of numerous aromatic secondary metabolites. The importance of this pathway in plants is indicated by the fact that about 20% of the carbon fixed by photosynthesis flow through this pathway. These facts strongly suggest that the modification of the shikimate and its derivative (or downstream) pathways may lead to dramatic changes in secondary metabolism. The enzymatic reactions in the biosynthetic pathway for aromatic amino acids seem to be identical in prokaryotes and eukaryotes but they apparently differ in the regulatory mechanisms for these pathways. For example, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the enzyme that catalyzes the first reaction in the shikimate pathway, is not feedback inhibited by any of the aromatic amino acids in plants, but, each of the three DAHP synthase isoenzymes are
Proceedings of the National Academy of Sciences, 2001
Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme⅐substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.