5-enol-Pyruvyl-Shikimate-3-Phosphate Synthase from Zea mays Cultured Cells (Purification and Properties) (original) (raw)
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Journal of Plant Physiology, 1997
Two isoforms of the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase, previously purified from maize cultured cells and both found to be functionally located in the plastid while showing a different pattern of expression during the culture growth cycle, were characterized with respect to physical and functional properties. A high degree of similarity was found as to their structural features, with the only exception of a slight difference in molecular mass. Both enzyme activities were extremely susceptible to the inhibition brought about by the herbicide glyphosate, and not subjected to feedback regulation by aromatic amino acids or shikimate pathway intermediates. A more pronounced difference was evident in thermal stability, catalytic efficiency as judged from the comparison of catalytic constants, affinities for the two substrates and activation energy values. The isozyme detectable in actively proliferating cells, when the plant cell demand for aromatic amino acids increases, proved to be the more stable and efficient. Data are consistent with the hypothesis of an isoform-based mechanism of enzyme level modulation in plant aromatic metabolism.
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, 1988
5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent K. values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (K, = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.
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
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.
Journal of …, 2011
The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme catalyses the first biosynthetic step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In plants the MEP pathway is involved in the synthesis of the common precursors to the plastidic isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate, in plastids. DXS is recognized as limiting this pathway and is a potential target for manipulation to increase various isoprenoids such as carotenoids. In Zea mays three dxs genes exist that encode plastid-targeted functional enzymes. Evidence is provided that these genes represent phylogenetically distinctive clades conserved among plants preceding monocot-dicot divergence. There is differential accumulation for each dxs gene transcript, during development and in response to external signals such as light. At the protein level, the analysis demonstrates that in Z. mays, DXS protein is feedback regulated in response to the inhibition of the pathway flow. The results support that the multilevel regulation of DXS activity is conserved in evolution.