Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

Mousdale, David M.; Coggins, John R.

doi:10.1007/bf00393514

Cited by 115 publications

(48 citation statements)

References 46 publications

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“…Other enzymes in the aromatic amino acid pathways, such as 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, 5-enolpyruvylshikimate-3-phosphate synthase, and tryptophan synthase A, have similar leader sequences and were shown biochemically to be present in chloroplasts (11,14,21,25).…”

Section: Discussionmentioning

confidence: 99%

A Phosphoribosylanthranilate Transferase Gene Is Defective in Blue Fluorescent Arabidopsis thaliana Tryptophan Mutants

Rose

Casselman

1992

Plant Physiol.

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Section: Discussionmentioning

confidence: 99%

A Phosphoribosylanthranilate Transferase Gene Is Defective in Blue Fluorescent Arabidopsis thaliana Tryptophan Mutants

Rose

Casselman

1992

Plant Physiol.

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“…Although Nicotiana silvestris is not closely related to V. radiata, a pair of isozymes similar to the V. radiata isozymes were noted in N. silvestris (7,8). This latter uniformity ofisozyme makeup is not reflected, however, by descriptions in the literature of DAHP synthase from corn (14), cauliflower (15), pea (20,22,23), tea (26), and carrot (28). Since we have found that assay conditions that favor the optimal detection of one isozyme are inadequate for detection of the second isozyme and vice versa, it is quite possible that this two-isozyme system may have escaped recognition in some other studies.…”

mentioning

confidence: 98%

Differentially Regulated Isozymes of 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Seedlings of Vigna radiata [L.] Wilczek

Rubin¹,

Jensen²

1985

Plant Physiol.

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ABSTRACIrTwo isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) designated DS-Mn and DS-Co were separated from seedlings of Vigna radiata [L.] Wilczek by DEAE-cellulose column chromatography. DS-Mn was activated 2.6-fold by 0.4 millimolar manganese, had an activity optimum of 7.0, and was substrate inhibited by erythrose-4phosphate (E4P) concentrations in excess of 0.5 millimolar. In contrast, DS-Co had an activity optimum at pH 8.8, required E4P concentrations of at least 4 millimolar to approach saturation, and exhibited an absolute requirement for divalent cation (cobalt being the best). Regulatory properties of the two isozymes differed dramatically. The activity of DS-Mn was activated by chorismate (noncompetitively against E4P and competitively against phosphoenolpyruvate), and was inhibited by prephenate and L-arogenate (competitively against E4P and noncompetitively against phosphoenolpyruvate in both cases). Under standard assay conditions, L-arogenate inhibited the activity of DS-Mn 50% at a concentration of 155 micromolar and was at least 3 times more potent than prephenate on a molar basis. Weak inhibition of DS-Mn by L-tryptophan was also observed, the magnitude of inhibition increasing with decreasing pH. The pattern of allosteric control found for DS-Mn is consistent with the operation of a control mechanism of sequential feedback inhibition governing overall pathway flux. DS-Co was not subject to aliosteric control by any of the molecules affecting DS-Mn. However, DS-Co was inhibited by caffeate (3,4-dihydroxycinnamate), noncompetitively with respect to either substrate. The striking parallels between the isozyme pairs of 3-deoxy-n.arabino-heptulosonate-7-phosphate synthase and chorismate mutase are noted-one isozyme in each case being tightly regulated, the other being essentially unregulated. 17, 1984, at Davis, CA. 2Abbreviations: DAHP, 3-deoxy-i-arabino-heptulosonate-7-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4phosphate. marins, indole derivatives, and other phenolic compounds (12).In mung bean we showed (25) that total DAHP synthase activity was distributed between two separable and quite distinctive isozymes. One isozyme (DS-Co) had an absolute requirement for divalent cation (cobalt being preferred). The second isozyme (DS-Mn) was stimulated by, but did not require, manganese. Although Nicotiana silvestris is not closely related to V. radiata, a pair of isozymes similar to the V. radiata isozymes were noted in N. silvestris (7,8). This latter uniformity ofisozyme makeup is not reflected, however, by descriptions in the literature of DAHP synthase from corn (14), cauliflower (15), pea (20,22,23), tea (26), and carrot (28). Since we have found that assay conditions that favor the optimal detection of one isozyme are inadequate for detection of the second isozyme and vice versa, it is quite possible that this two-isozyme system may have escaped recognition in some other studies. For example, although a single enzyme that appears similar to DS-Mn wa...

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“…1) indicates that the mature enzyme is organelle-bound. Evidence has accumulated over the past years that EPSP-synthase, as well as the entire shikimate pathway, functions in the plastid (see Mousdale and Coggins [12] for references). The localization of EPSP-synthase in the plastids of cultured C. sempervirens cells has been confirmed by protein A-gold immunocytochemistry (CC Smart, N Amrhein, unpublished data) as well as cell fractionation studies (J Johanning, N Amrhein, unpublished data).…”

mentioning

confidence: 99%

5-Enolpyruvylshikimate 3-Phosphate Synthase, the Target Enzyme of the Herbicide Glyphosate, Is Synthesized as a Precursor in a Higher Plant

Holländer-Czytko

Amrhein²

1987

Plant Physiol.

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Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30-to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (M,) 53900 ± 900 as compared to M, 45500 ± 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures.The nonselective, broad spectrum herbicide glyphosate, N-(phosphonomethyl)glycine, has been shown to be a potent and efficient inhibitor of the shikimate pathway enzyme EPSPsynthase2 from plants and microorganisms (1,4,11,22 (Sigma) were used for precipitation with either preimmune serum (5 Ml) or the antiserum (20 Ml). The latter had been affinity-purified using EPSP-synthase bound to nitrocellulose (A. Schulz, personal communication). Radioactive proteins were eluted from the protein A complex and analyzed by SDS-gel electrophoresis on a 8 to 13% polyacrylamide gel (9). The gel was incubated with Enhance (NEN), dried, and exposed to a Kodak XAR5 film. Fluorograms were photographed, printed on an Agfaortho 25 document copying film, and scanned at 620 nm with an Isco gel scanner for quantification. For analysis of immunoprecipitable material by SDS-gel electrophoresis, between 2 and 4 translation assays were combined. About 0.1% of radioactivity incorporated into protein could be recovered in the immunoprecipitates from assays containing RNA from nonadapted cells as compared to 0.8 to 1% from adapted cells (Table I). Further analysis by SDS-electrophoresis and fluorography revealed that the immunoprecipitable material was concentrated in a single band of Mr 53900 ± 900 (mean ± SD of 7 independent experiments) ( Fig. 1). As precipitation with preimmune serum produced no radioactive band (data not shown) and as affinity-purified anti4EPSP-synthase) serum was employed, we conclude that the band represents a higher mol wt precursor of EPSP-synthase, the mature form of which has a Mr of 45,500 ± 1,000 (20

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Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

Cited by 115 publications

References 46 publications

A Phosphoribosylanthranilate Transferase Gene Is Defective in Blue Fluorescent Arabidopsis thaliana Tryptophan Mutants

A Phosphoribosylanthranilate Transferase Gene Is Defective in Blue Fluorescent Arabidopsis thaliana Tryptophan Mutants

Differentially Regulated Isozymes of 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Seedlings of Vigna radiata [L.] Wilczek

5-Enolpyruvylshikimate 3-Phosphate Synthase, the Target Enzyme of the Herbicide Glyphosate, Is Synthesized as a Precursor in a Higher Plant

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