3,324 research outputs found
Differential expression of 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms in elicitor-treated, cultured maize cells
No full textThe 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
Purification and properties of a pyruvate carboligase from Zea mays cultured cellsfn1fn1Part 2 in the series Acetoin synthesis in higher plants.
No full textAn enzyme able to catalyze the synthesis of acetoin (3-hydroxy-2-butanon) from either pyruvate or acetaldehyde was isolated, partially purified and characterized from maize (Zea mays L. cv Black Mexican Sweet) cultured cells. It exhibited a maximal rate at neutral pH values, and strictly required thiamine pyrophosphate and a divalent cation for activity; on the contrary, unlike bacterial pyruvate oxidases, flavin was not required. Apparent Michaelis constants were 260 +/- 20 micromolar for pyruvate and 24 +/- 7 micromolar for acetaldehyde. Both substrate affinity and specificity were notably higher than those of pyruvate decarboxylase, an enzyme that also synthesizes acetoin as by-product. The partially purified protein was unable to catalyze the formation of other possible products of pyruvate decarboxylation, thus carboligase appears to be its main activity. Results suggest that acetoin synthesis may be of physiological significance in plants
Purification and properties of a cytosolic glutamine synthetase expressed in Nicotiana plumbaginifolia cultured cells
No full textGlutamine synthetase (EC 6.3.1.2) was purified and characterized from leaf protoplast-derived suspension cultured cells of Nicotiana plumbaginifolia. Maximal specific activity observed reflected a purification of over 1 500-fold, with a yield of about 40%. The native protein appeared to be an octamer, with subunits of a molecular mass of 37 kDa. Subcellular fractionation experiments accounted for a cytosolic localization of the enzyme. No evidence for multiple enzyme forms was found following either anion-exchange chromatography or native polyacrylamide gel electrophoresis. Results also suggest that in the absence of intercellular transport, type-1 glutamine synthetase may function preferentially to assimilate ammonia from primary nitrate reduction
Effects of Substituent and Scaffold Changes on the Inhibition of Human P5C Reductase by Phenyl-Substituted Aminomethylene Bisphosphonates
No full textBeing overexpressed in many cancer types and related to tumor invasiveness, the activity of P5C reductase represents a promising target for cancer therapy, yet no effective inhibitors have been identified so far. Several phenyl-substituted aminomethylenebisphosphonic acids had been found to inhibit the plant enzyme in the micro- to millimolar range. The two most active compounds were previously shown to be remarkably active against human P5C reductase (PYCR1, gene ID 5831). To investigate their structure–activity relationships, the human enzyme was heterogously expressed in E. coli, affinity purified and assayed in the presence of increasing concentrations of various aminobisphosphonates differing in substituents on the phenyl ring, using either NADH or NADPH as the electron donor. Some analogues, namely aminoethylenebisphosphonates, hydroxybisphosphonates, aminophosphonates and hydroxyphosphonates, were also evaluated. Results allowed to define the chemical features required for effective inhibition. The aminobisphosphonic moiety was found essential for activity, which was enhanced by the presence of electron-withdrawing substituents on the phenyl ring, provided that an optimal steric hindrance is not exceeded. These results could open up new perspectives on the synthesis of effective inhibitors of human P5C reductase to be used in chemotherapy
Differential in vitro responses of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea. 2. Aromatic biosynthesis.
No full textPhenylalanine ammonia lyase specific activity levels were measured in suspension cultured cells of six rice cultivars following the treatment with cell wall hydrolysates prepared from seven Pyricularia grisea strains. Early after elicitation, even low hydrolysate concentrations were able to induce a significant increase of enzyme levels. However, neither rice genotypes showing differential sensitivity to blast reacted differently, nor elicitors obtained from various pathotypes induced different reactions. At a later stage, higher hydrolysate concentrations were required to trigger maximal enzyme induction. In this case also, only slight variations were detected in suspension cultures of a given cultivar treated with different elicitors. On the contrary, highly significant differences were disclosed among plant genotypes. A remarkable relationship was evident between the mean increase in phenylalanine ammonia lyase activity and the overall resistance to blast at the plant level. This trait could therefore represent a useful tool aiming at the selection for increased blast tolerance
Properties of the 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms isolated from maize cultured cells
No full textTwo 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 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 feed-back regulation by aromatic amino acids or shikimate pathway intermediates. A more pronounced difference was evident in thermal stability, and 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, resulted the more stable and efficient. Data are consistent with the hypothesis of an isoform-based mechanism of enzyme level modulation in plant aromatic metabolism
Toward Unveiling the Mechanisms for Transcriptional Regulation of Proline Biosynthesis in the Plant Cell Response to Biotic and Abiotic Stress Conditions
No full textProline accumulation occurs in plants following the exposure to a wide array of stress conditions, as well as during numerous physiological and adaptive processes. Increasing evidence also supports the involvement of proline metabolism in the plant response to pathogen attack. This requires that the biosynthetic pathway is triggered by components of numerous and different signal transduction chains. Indeed, several reports recently described activation of genes coding for enzymes of the glutamate pathway by transcription factors (TFs) belonging to various families. Here, we summarize some of these findings with special emphasis on rice, and show the occurrence of a plethora of putative TF binding sites in the promoter of such genes
The role of proline in the adaptation of eukaryotic microalgae to environmental stress: An underestimated tool for the optimization of algal growth
No full textMicroalgae are considered the most promising source of renewable fuels, high-value bio-products and nutraceuticals. Potentially, microalgae can satisfy many global demands, but in large-scale cultivation the average productivity of most industrial strains is lower than maximal theoretical estimations, mainly due to sub-optimal growth conditions. Although microalgae have developed complex strategies to cope with environmental stresses, cultivation in outdoor photobioreactors is limited to few species and it is not yet sufficiently remunerative. Indeed, most microalgal species are very sensitive to environmental conditions, and changes in solar irradiation, temperature, and medium composition can drastically decrease biomass yield. Developing new strategies for improving algal tolerance to stress conditions is thus greatly desirable. One of the first responses that occur in both higher plants and microorganisms following the exposure to abiotic stress conditions, is an increased synthesis and accumulation of the amino acid proline. While the role of proline accumulation in stress adaptation is well-recognized in higher plants, in microalgae the implication of proline in stress tolerance still awaits full elucidation. In this review we summarize available data on proline metabolism under environmental stress in eukaryotic microalgae. Possible implications toward optimization of algal growth for biotechnological purposes are also discussed
A dimeric 5-enol-pyruvyl-shikimate-3-phosphate synthase from the cyanobacterium Spirulina platensis
No full textIsolation and biochemical characterization is reported here of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis.
Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation.
A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phosphoenolpyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa.
The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono- and dimeric EPSP synthases could represent an important tool for cyanobacterial classification
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