L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes

Gaines, C G; Byng, Graham S.; Whitaker, Robert J.; Jensen, Roy A.

doi:10.1007/bf00393730

Cited by 75 publications

(55 citation statements)

References 25 publications

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“…A similar assumption prevailed for microorganisms until 1974, when enzymatic formation of L-arogenate from prephenate and enzymatic conversion of L-arogenate to L-tyrosine was first recognized in cyanobacteria (2). Since then, the arogenate pathway to L-tyrosine has been demonstrated in mung bean (3), corn (4), sorghum (5), tobacco (6), spinach (7), and buckwheat (J. L. Rubin and R.A.J., unpublished data). Prephenate dehydrogenase activity has not been found in any plant system, except in the developmental stage of seed germination in mung bean.…”

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confidence: 74%

Chloroplasts of higher plants synthesize L-phenylalanine via L-arogenate.

Jung¹,

Zamir²,

Jensen³

1986

Proc. Natl. Acad. Sci. U.S.A.

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The specific enzymological route of L-phenylalanine biosynthesis has not been established in any higher plant system. The possible pathway routes that have been identified in microorganisms utilize either phenylpyruvate or L-arogenate as a unique intermediate. We now report the presence ofarogenate dehydratase (which converts L-arogenate to L-phenylalanine) in cultured-cell populations of Nicotiana silvestris. Prephenate dehydratase (which converts prephenate to phenylpyruvate) was not detected. Arogenate dehydratase was also found in washed spinach chloroplasts, and these data add to emerging evidence in support of the existence in the plastidial compartment of a complete assembly of enzymes comprising aromatic amino acid biosynthesis. Arogenate dehydratase from tobacco and spinach were both specific for L-arogenate, inhibited by L-phenylalanine, and activated by L-tyrosine. Apparent K. values for L-arogenate (0.3 x 10-3 M), pH optima (pH 8.5-9.5), and temperature optima for catalysis (32-340C) were also similar.The current understanding of the post-prephenate pathways of phenylalanine and tyrosine biosynthesis and of their regulation has depended almost entirely upon a large base of comparative data from both prokaryotic and eukaryotic microorganisms (1). Until recently it had been assumed that higher plants utilize phenylpyruvate and 4-hydroxyphenylpyruvate as biosynthetic precursors of L-phenylalanine and L-tyrosine, respectively. A similar assumption prevailed for microorganisms until 1974, when enzymatic formation of L-arogenate from prephenate and enzymatic conversion of L-arogenate to L-tyrosine was first recognized in cyanobacteria (2). Since then, the arogenate pathway to L-tyrosine has been demonstrated in mung bean (3), corn (4), sorghum (5), tobacco (6), spinach (7), and buckwheat (J. L. Rubin and R.A.J., unpublished data). Prephenate dehydrogenase activity has not been found in any plant system, except in the developmental stage of seed germination in mung bean. Hence, the arogenate pathway appears to be the major, if not exclusive, mode of L-tyrosine biosynthesis in higher plants.Progress with the phenylalanine pathway has been slower, largely because ofthe technical difficulty ofthe enzyme assay (8,9). Fig. 1 (Binghamton, NY). The background and characteristics of the cell line ANS-1 of N.silvestris have been described (6). Suspension cultures of these cells were maintained by 1:4 dilution every 8 days into fresh medium. In this subculture routine, the cells undergo a lag phase of "'36 hr followed by a period of exponential growth that lasts until about day 6, when the cells enter a stationary phase.Cells were harvested in late-exponential phase between days 4 and 5 on Miracloth filters and were washed three times with 3% (wt/vol) mannitol. Excess wash solution was removed with the aid of a suction filter. The cell pack was frozen in liquid nitrogen and stored at -80'C or was ground in liquid nitrogen using a Waring blender and stored as a frozen powder at -80'C.Preparation of Partially...

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confidence: 74%

Chloroplasts of higher plants synthesize L-phenylalanine via L-arogenate.

Jung¹,

Zamir²,

Jensen³

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The second isozyme (DS-Mn) was stimulated by, but did not require, man-ganese. 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).…”

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confidence: 95%

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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“…Two isozymes of DAHP synthase were identified in Vigna radiata (23,24), and were denoted DAHP synthase-Mn (stimulated by, but not requiring Mn) and DAHP synthase-Co (requiring Co, Mg, or Mn for activity). This pair of DAHP synthase isozymes was also isolated from leaves of N. silvestris (9). When assay conditions were optimized for either isozyme, the activity of the remaining isozyme was barely detectable.…”

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confidence: 99%

The Two-Isozyme System of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Nicotiana silvestris and Other Higher Plants

Ganson¹,

d'Amato²,

Jensen³

1986

Plant Physiol.

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Two isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, denoted DS-Mn and DS-Co, were identified following DEAE-cellulose chromatography of crude extracts prepared from suspension-cultured cells of Nicotiana silvestris. The strikingly different properties of the isozymes allowed the development of assays for the selective detection of either isozyme in samples containing a mixture of the two. The DS-Mn isozyme required the sulfhydryl reductant, dithiothreitol, for activity and was stimulated by manganese. Activation by dithiothreitol was slow relative to catalysis, accounting for a hysteretic progress curve that was observed when reactions were started with inactive enzyme. The DS-Co isozyme was inhibited by dithiothreitol and required a divalent cation for activity. At optimal cation concentrations of 10 millimolar (magnesium), 0.5 millimolar (cobalt), and 0.5 millimolar (manganese), relative activities obtained were 100, 85, and 20, respectively. The substrate saturation curves with respect to erythrose 4-phosphate differed markedly when the two isozymes were compared. As little as 0.5 millimolar erythrose 4-phosphate saturated DS-Mn, whereas a 10-fold higher concentration was needed for saturation of DS-Co. The pH optimum of DS-Mn was 8.0, while that of the DS-Co isozyme was 8.6. Leaves of both N. silvestris and spinach also exhibited the DS-Mn/ DS-Co isozyme arrangement, and the subcellular location of DS-Mn was shown to be the chloroplast compartment. By application of the differential assays for DAHP synthase isozymes, various monocotyledonous and dicotyledonous plants yielded data indicating the general presence of the DS-Mn/DS-Co isozyme pair in higher plants.biosynthesis and the separate subcellular location of such isozymes is just developing (16,17). The best studied example is chorismate mutase, shown in Nicotiana silvestris to exist as two isozymes (11,12): an allosterically controlled species (CM-1) being located in plastids and an unregulated species (CM-2) being located in the cytosol (7). Two isozymes of DAHP synthase were identified in Vigna radiata (23,24), and were denoted DAHP synthase-Mn (stimulated by, but not requiring Mn) and DAHP synthase-Co (requiring Co, Mg, or Mn for activity). This pair of DAHP synthase isozymes was also isolated from leaves of N. silvestris (9). When assay conditions were optimized for either isozyme, the activity of the remaining isozyme was barely detectable.The markedly distinct properties of DS-Mn and DS-Co from N. silvestris have been exploited to define assay conditions for exclusive assay of either one of the two isozymes in mixtures. A reliable methodology to discriminate DAHP synthase isozymes in crude extracts is particularly valuable for subcellular localization studies as well as assessment of the variation in isozyme levels in response to developmental and regulatory changes. Although DAHP synthase from a variety of plants has been studied (14,15,(20)(21)(22)26), particular assay conditions employed would have favored in mo...

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L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes

Cited by 75 publications

References 25 publications

Chloroplasts of higher plants synthesize L-phenylalanine via L-arogenate.

Chloroplasts of higher plants synthesize L-phenylalanine via L-arogenate.

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

The Two-Isozyme System of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Nicotiana silvestris and Other Higher Plants

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