Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycitie], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzynies, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of commonpathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry of energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coi, after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also produced profound effects upon glyphosate sensitivity.Many antimetabolites that act against biosynthetic pathways mimic end products, causing inappropriate regulatory effects upon enzymes having early-pathway positions. In contrast, N-(phosphonomethyl)glycine (PMG), an herbicide known as glyphosate, is an exceedingly effective inhibitor of a mid-pathway enzyme of aromatic amino acid biosynthesis, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase. EPSP synthase has been shown to be a sensitive target of PMG action in procaryotes, lower eucaryotes, and higher plants (see reference 34 for reference citations). Although PMG is now under intensive study in higher plants by virtue of its potent herbicidal properties, antimicrobial studies are few. The presence of PMG during growth c...
The biosynthetic route to L-tyrosine was identified in isogenic suspension-cultured cells of N. silvestris. Arogenate (NADP(+)) dehydrogenase, the essential enzyme responsible for the conversion of L-arogenato L-tyrosine, was readily observed in crude extracts. In contrast, prephenate dehydrogenase (EC 1.3.1.13) activity with either NAD(+) or NADP(+) was absent altogether. Therefore, it seems likely that this tobacco species utilizes the arogenate pathway as the exclusive metabolic route to L-tyrosine. L-Tyrosine (but not L-phenylalanine) was a very effective endproduct inhibitor of arogenate dehydrogenase. In addition, analogs of L-tyrosine (m-fluoro-DL-tyrosine [MFT], D-tyrosine and N-acetyl-DL-tyrosine), but not of L-phenylalanine (o-fluoro-DL-phenylalanine and p-fluoro-DL-phenylalanine), were able to cause inhibition of arogenate dehydrogenase. The potent antimetabolite of L-tryptophan, 6-fluoro-DL-tryptophan, had no effect upon arogenate dehydrogenase activity. Of the compounds tested, MFT was actually more effective as an inhibitor of arogenate dehydrogenase than was L-tyrosine. Since MFT was found to be a potent antimetabolite inhibitor of growth in N. silvestris and since inhibition was specifically and effectively reversed by L-tyrosine, arogenate dehydrogenase is an outstanding candidate as the in vivo target of analog action. Although chorismate mutase (EC 5.4.99.5) cannot be the prime target of MFT action, MFT can mimick L-tyrosine in partially inhibiting this enzyme activity. The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was insensitive to L-phenylalanine or L-tyrosine. The overall features of this system indicate that MFT should be a very effective analog mimick for selection of feedback-insensitive regulatory mutants L-tyrosine biosynthesis.
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. [271, and tobacco [unpublished data]) are now coming to light. Second, the particular details of reversal of glyphosate inhibition by aromatic acids are variable from organism to organism. In E. coli, all three aromatic amino acids are required for complete reversal (25). In Rhizobium japonicum, the combination of phenylalanine plus tyrosine reversed glyphosate inhibition (18) in contrast to Lemna gibba where phenylalanine alone was adequate for reversal (18). In Euglena gracilis, aromatic amino acids only partially reverse inhibition (40%o) unless the minor pathway products: 4-aminobenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, and 3,4-dihydroxybenzaldehyde are also present (5).Where partial reversal of inhibition is obtained with either Lphenylalanine alone or L-tyrosine alone, the greatest reversal is usually accomplished by L-phenylalanine. For example, the antagonism of inhibition by L-phenylalanine and L-tyrosine was 41% and 12% in Escherichia coli, and 27% and 3% in Chlamydomonas reinhardii (15). Qualitatively, it was found that L-phenylalanine was a better partial antagonist of glyphosate than L-tyrosine in suspension cultures of both carrot and soybean (15)
Treatment of isogenic suspension-cultured cells of Nicotiana silvestrisSpeg. et Comes with glyphosate (N-phosphonomethyljglycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-D-arabiaoheptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ionexchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK. values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO-CH2NH2+CH2PO32-, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K,' = 183 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an lenzyme:shikimate-3-PJ complex and ultimately forms the dead-end complex of lenzyme:shikimate-3-P:glyphosatel.Glyphosate (N-[phosphonomethyl]glycine) is a broad-spectrum herbicide which is also a potent growth inhibitor ofbacteria and algae (14). Although glyphosate may ultimately perturb a variety of biochemical processes including protein synthesis, nucleic acid synthesis, photosynthesis and respiration (16), the early conjecture (18)
The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by L-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by L-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.
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