Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris

Bonner, Carol A.; Jensen, Roy A.

doi:10.1016/0003-9861(85)90161-4

Cited by 46 publications

(40 citation statements)

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“…The possibility that the latter activity may be present only during the developmental time of seedling germination is being examined. The transamination of prephenate is carried out by [AT-C], an aminotransferase previously denoted PPA-AT in mung bean (24), and similar to the recently characterized aminotransferase activity in N. silvestris (1). CM-I is an isozyme of chorismate mutase that is activated by L-tryptophan and feedback inhibited by L-tyrosine and Lphenylalanine in mung bean (10).…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…L-Arogenate is already known to be at least a major precursor of L-tyrosine in various higher plant species (3-7). A highly active aminotransferase enzyme having an unusually narrow substrate specificity for prephenate exists in mung bean (3), tobacco (23), and sorghum (C. A. Bonner and R.A.J., unpublished data). L-Arogenate plays a role as a feedback inhibitor of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase-Mn) in the chloroplast (24,25).…”

Section: Resultsmentioning

confidence: 99%

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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“…Purified plant aromatic aminotransferases possess properties similar to most animal and microbial aminotransferases. Several reports have suggested that plant Trp and Asp aminotransferases have the same substrate multispecificity (Bonner and Jensen, 1985) as mammalian and microbial Asp aminotransferases (Mavrides and Orr, 1975). Plant Asp aminotransferases have been shown to transaminate five L-amino acids, Asp, Glu, Phe, Tyr, and Trp, using a-ketoglutarate or oxaloacetate as the amino group acceptor (Forest and Wightman, 1972).…”

Section: Involvement Of Tyrat In Bia Metabolism In Opium Poppymentioning

confidence: 99%

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

Lee

Facchini

2011

Plant Physiology

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Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and a-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5#-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and a-ketoglutarate, with apparent K m values of 1.82 and 0.35 mM, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

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Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris

Cited by 46 publications

References 19 publications

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

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

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

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

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