EPSP Synthase

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g−1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5‐enol‐pyruvyl‐shikimate‐3‐phosphate synthase, the target site of glyphosate action, was highly sen...

The large-scale use of the herbicide glyphosate leads to growing ecotoxicological and human health concerns. Microbe-assisted phytoremediation arises as a good option to remove, contain, or degrade glyphosate from soils and waterbodies, and thus avoid further spreading to non-target areas. To achieve this, availability of plant-colonizing, glyphosate-tolerant and -degrading strains is required and at the same time, it must be linked to plant-microorganism interaction studies focusing on a substantive ability to colonize the roots and degrade or transform the herbicide. In this work, we isolated bacteria from a chronically glyphosate-exposed site in Argentina, evaluated their glyphosate tolerance using the minimum inhibitory concentration assay, their in vitro degradation potential, their plant growth-promotion traits, and performed whole genome sequencing to gain insight into the application of a phytoremediation strategy to remediate glyphosate contaminated agronomic soils. Twenty-...

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g À1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5-enol-pyruvyl-shikimate-3-phosphate synthase, the target site of glyphosate action, was highly sensitive to the herbicide. Fungal growth rate was considerably lower when the herbicide was also the only phosphorus source, whereas glyphosate utilization was substantially unaffected, suggesting an unusual route for its degradation. Herbicide metabolism was strongly reduced when other sources of organic nitrogen were made available.

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.

Possible non-target effects of the widely used, nonselective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.

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.

Summary As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5‐enol‐pyruvyl‐shikimate‐3‐phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering sep...

Possible non-target effects of the widely used, nonselective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g−1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5‐enol‐pyruvyl‐shikimate‐3‐phosphate synthase, the target site of glyphosate action, was highly sen...

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.

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.

Possible non-target effects of the widely used, nonselective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.

A Penicillium chrysogenum strain was isolated for its ability to grow in minimal medium containing the herbicide glyphosate as the only nitrogen source. The presence of concentrations up to 25 mM progressively stimulated the fungal growth rate, which was negligible in media lacking reduced nitrogen. However, glyphosate utilization never exceeded 1 mmol g À1 mycelial dry mass, and below a threshold concentration both herbicide uptake and fungal growth were subject to a lag phase, suggesting that the herbicide may enter the cell by either simple passive diffusion or inducible carriers. Amino acids, possible products of glyphosate breakdown, as well as ammonia, were found to replace the herbicide in restoring mycelial growth. Cells were devoid of detectable nitrate reductase activity, thus the isolate seems to be impaired in its ability to convert nitrate to ammonium. In vitro activity of 5-enol-pyruvyl-shikimate-3-phosphate synthase, the target site of glyphosate action, was highly sensitive to the herbicide. Fungal growth rate was considerably lower when the herbicide was also the only phosphorus source, whereas glyphosate utilization was substantially unaffected, suggesting an unusual route for its degradation. Herbicide metabolism was strongly reduced when other sources of organic nitrogen were made available.

Flexibility is involved in a wide range of biological processes, such as protein assembly and binding recognition. EPSP synthase is an enzyme that must undergo a large con-formational change to accommodate its ligands into its binding cavity. However, although the structure of EPSP synthase has been determined, its plasticity has not been explored in depth. Therefore, in this work, we extensively examined the influence of the flexibility of Mycobacterium tuberculosis EPSP (MtEPSP) synthase on the function of this protein using classical and replica-exchange metadynamics simulations. We were able to identify five well-populated conformational clusters for MtEPSP synthase: two corresponding to open, one to ajar, and two to closed conformations. We also pinpointed three hydrophobic regions that are responsible for guiding transitions among these states. Taken together, the new findings presented here indicate how the hydrophobic regions modulate the flexibility of MtEPSP synthase, and they highlight the importance of considering these dynamic features in drug design projects employing this enzyme as a target.