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N-pyridyl-aminomethylene-bisphosphonic acids inhibit the first enzyme in the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase
No full textA series of seven structurally diverse N -substituted aminomethylene bisphosphonic acids exhibiting remarkable herbicidal activity was found to inhibit the activity of the first enzyme in the prechorismate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, partially purified from Nicotiana plumbaginifolia suspension cultured cells. At millimolar concentrations all compounds inhibited the Co 2+ -dependent, cytosol-localized enzyme form. However, the addition of excess divalent cations to the reaction mixture was able to completely relieve their effect, suggesting that the chelating properties of these compounds could account for their inhibitory effect. In contrast, only five compounds of seven reduced significantly the activity of the plastidial and Mn 2+ -stimulated isoform. The inhibition brought about by N -2-(6-methyl-pyridyl)- and N -2-(5-chloro-pyridyl)-aminomethylene bisphosphonic acid could not be relieved by raising the manganese concentration in the assay mixture. A kinetic analysis showed that the latter compound inhibits enzyme activity uncompetitively with respect to phospho enol pyruvate, competitively with respect to the other substrate, erythrose-4-phosphate. As manganese is a V max stimulator of the plastidial enzyme, this ruled out the possibility of an inhibition simply based upon metal chelation
A metal-independent hydrolase from a Penicillium oxalicum strain able to use phosphonoacetic acid as the only phosphorus source.
No full textA Penicillium oxalicum strain was capable of the phosphate-sensitive utilization of phosphonoacetic acid as the sole source of phosphorus. A carbon-to-phosphorus bond-cleavage enzyme yielding acetic acid and inorganic phosphate was detected and characterized in extracts from cells grown on this phosphonate. Contrary to bacterial phosphonoacetate hydrolases, the fungal enzyme neither required nor was stimulated by divalent cations
The herbicidally active compound N-2-(6-methyl-pyridyl)-aminomethylene bisphosphonic acid inhibits in vivo aromatic biosynthesis.
No full textThe effect of N-2-(6-methyl-pyridyl)-aminomethylene bisphosphonic acid (M-pyr-AMBPA), a compound previously shown to exhibit herbicidal properties on whole plants and to inhibit in vitro activity of the first enzyme in the shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, was investigated on Nicotiana plumbaginifolia suspension cultured cells and compared to that of the herbicide glyphosate. The addition of M-pyr-AMBPA from 10-4 to 10-3 M was found to cause a severe cell growth reduction. Kinetic analysis of partially purified DAHP synthase accounted for non-competitive inhibition type with respect to both phospho-enol-pyruvate and erythrose-4-phosphate, with KI values of 0.43 and 0.62 mM, respectively. Amino acid pool measurements of cells grown in the presence of sublethal doses of M-pyr-AMBPA pointed to an actual reduction of free aromatic amino acids, showing that DAHP synthase inhibition takes place in vivo, and suggesting that the interference of this aminophosphonate with plant aromatic biosynthesis may account for a large part of its phytotoxicity. However, exogenous supply of a mixture of phenylalanine, tyrosine and tryptophan failed to achieve full reversal of cell growth inhibition, yet the occurrence of other target(s) cannot be ruled out
The herbicidally active compound N-2-(5-chloro-pyridyl) aminomethylene bisphosphonic acid acts by inhibiting both glutamine and aromatic amino acid biosynthesis
No full textThe effect of the herbicidally active compound N-2-(5-chloro-pyridyl)aminomethylene bisphosphonic acid (Cl-pyr-AMBPA), previously found in vitro to inhibit the activity of the first enzyme in the shikimate pathway 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, was investigated in vivo on suspension cultured cells of Nicotiana plumbaginifolia Viviani. Amino acid pool measurement showed an actual reduction of tyrosine, tryptophan and phenylalanine level following the addition of the compound to the growth medium. However, an even stronger effect was noticed for other amino acids, mainly glutamine. When the activity of the enzymes involved in the glutamate cycle was measured in the presence of Cl-pyr-AMBPA, glutamate synthase was unaffected, while glutamine synthetase was significantly inhibited. Contrary to the herbicide phosphinothricin, the inhibitor bound reversibly to the enzyme. Kinetic analysis accounted for an inhibition of uncompetitive type with respect to ammonium, glutamate and ATP, withKivalues of 113, 97 and 39 M, respectively. Only the exogenous supply of a mixture of glutamine and aromatic amino acids relieved cell growth inhibition, suggesting that the phytotoxic properties of Cl-pyr-AMBPA are due to inhibition of key enzymes in both the corresponding pathways
Isolation and characterization of two new microbial strains capable of degradation of the naturally occurring organophosphonate–ciliatine
No full textAir-born mixed fungal and bacterial culture capable of complete degradation of ciliatine was isolated. The utilization of the natural organophosphonate proceeded in the phosphate independent manner. Enzymatic activity involved in ciliatine degradation studied in the fungal cellfree extract proved to be distinct from bacterial
pathway described before
Metabolism of the phosphonate herbicide glyphosate by a non-nitrate-utilizing strain of Penicillium chrysogenum
No full textA 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 I 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 weft 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
Phosphonoacetate hydrolase from Penicillium oxalicum: purification and properties, phosphate starvation-independent expression, and partial sequencing.
No full textThe enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed
Mode of action of herbicidal derivatives of aminomethylenebisphosphonic acid. II. Reversal of herbicidal action by aromatic amino acids.
No full textThe herbicidal action of N-pyridylaminomethylenebisphosphonic acids is accompanied by an impairment of anthocyanin biosynthesis. This suggests that they might act as inhibitors of some steps in aromatic amino acid biosynthesis. Herbicidal effects were reversed by aromatic amino acids using both bacterial and plant models, a finding that strongly supports this hypothesis. Structural features of these compounds suggest the sixth enzyme in the shikimate pathway 5-enol-pyruvoylshikimate-3-phosphate (EPSP) synthase as a possible target, since a strong structural similarity exists between aminomethylenebisphosphonic acid and an inhibitor of EPSP synthase, the herbicide glyphosate. This is, however, not the case since they did not act as inhibitors of this enzyme
Herbicidal pyridyl derivatives of aminomethylene-bisphosphonic acid inhibit plant glutamine synthetase
No full textA series of aminomethylene-bisphosphonic acid derivatives, previously synthesized and shown to be endowed with herbicidal properties, were evaluated as potential inhibitors of plant glutamine synthetase. The cytosolic form of the enzyme was partially purified from rice cultured cells and assayed in the presence of millimolar concentrations of the compounds by means of three different assay methods, respectively measuring the hemibiosynthetic, the transferase, and the full biosynthetic reactions. Several compounds were found to exert a remarkable inhibition, with I50 values similar to those obtained under the same conditions with a well-established inhibitor of glutamine synthetase, the herbicide phosphinothricin. Contrary to the reference compound, enzyme kinetics accounted for a reversible inhibition mechanism. The biological activity of the most active derivatives was further characterized by measuring free glutamine levels in cell suspension rice cultures following treatment with the inhibitors. Results confirmed their ability to interfere in vivo with nitrogen metabolism. A preliminary analysis of structure-activity relationship allowed it to be hypothesized that steric rather than electronic factors are responsible for the inhibitory potential of these compounds
Herbicidal derivatives of aminomethylenebisphosphonic acid. Part III. Structure-activity relationship
No full textDerivatives of aminomethylenebisphosphonic acids constitute a class of promising herbicides. More than 40 N-substituted aminomethylenephosphonic acids were synthesized and evaluated for their herbicidal activity on common cress (Lepidium sativum L.) and cucumber (Cucumis sativus L.). Some of the tested compounds were found to exhibit strong herbicidal properties being equal in activity with the popular herbicide glyphosate as well as parent N-pyridylaminomethylenephosphonic acids. N-Substituted iminodi(methylenephosphonic) acids, which may be considered as close analog of glyphosate, were inactive toward test plants
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