Purification and characterization of anthranilate synthase from <i>Catharanthus roseus</i>

Poulsen, Charlotte; Bongaerts, Roger J. M.; Verpoorte, Robert

doi:10.1111/j.1432-1033.1993.tb17679.x

Cited by 73 publications

(48 citation statements)

References 36 publications

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“…The purified GST-ASA1 exhibited enzymatic properties similar to those observed for AS purified from C. roseus (Poulsen et al, 1993). The apparent KM for chorismate was about 180 p~ in the presence of 100 m~ ammonium chloride, in accordance with reported values (Table I).…”

Section: Optimization Of Active Gst-asa1 Isolationsupporting

confidence: 88%

“…The sensitive form was found to be predominant in normal cells, and the insensitive form was found in 5-methyltryptophan-resistant lines. In contrast, Poulsen et al (1993) found no evidence for a Trpinsensitive form of AS in C. roseus. However, they separated two sensitive forms of AS from the crude extract in an anionexchange column.…”

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confidence: 74%

“…AS from plants has not been purified to homogeneity. The only extensively purified AS is from the cell cultures of Catharanthus roseus (Poulsen et al, 1993). After five purifi-* Corresponding author; fax 1-415-493-1073. cation steps, including four involving column chromatography, 54 pg of AS was obtained from 1 kg of cells, with a specific activity of 164 nmol min-' mg-*.…”

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confidence: 99%

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Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

Bernasconi¹,

Walters²,

Woodworth³

et al. 1994

Plant Physiol.

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Anthranilate synthase is involved in tryptophan (Tip) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione Stransferase (CST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-' mg-'), the apparent K, for chorismate (180 &M), and the feedback inhibition by Trp (complete inhibition by 10 f i~ Trp) of the purified fusion product (CST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion protein product and purified by affinity chromatography on a CST-ASAl-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. CST-ASA1 cleavage by thrombin, as well as sitedirected mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.

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Section: Optimization Of Active Gst-asa1 Isolationsupporting

confidence: 88%

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confidence: 74%

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confidence: 99%

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Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

Bernasconi¹,

Walters²,

Woodworth³

et al. 1994

Plant Physiol.

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“…Plants show a similarly complex regulation. Feedback regulation of both enzyme activities has been reported in various plants as well (Figure 2) (Poulsen et al 1993;Li and Last 1996;Goers and Jensen 1984;Mobley et al 1999). In addition, the expression of the genes encoding AS and CM is also regulated at the transcription stage.…”

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confidence: 78%

Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice

Wakasa

Ishihara

2009

Plant Biotechnology

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Tryptophan (Trp) and phenylalanine (Phe) are essential aromatic amino acids that animals cannot synthesize and are dependent on plants for their supply. In particular, Trp contributes to the nutritional quality of plant-based foods, and, along with lysine, methionine and threonine, it is used to supplement animal feeds. The other aromatic amino acids, Phe and tyrosine (Tyr), are used for the production of the low-calorie sweetener aspartame and the anti-Parkinson's disease drug L-dopa, respectively. The biosynthetic pathways for aromatic amino acids and their regulation have been extensively explored in bacteria because of their utility in the food and drug industry. In plants, aromatic amino acids are the precursors for a large variety of secondary metabolites including indole alkaloids, phenylpropanoids, flavonoids, and the phenolic polymer, lignin. One of plant growth regulators, indole-3-acetic acid (IAA), has also been shown to be biosynthesized from Trp. Thus, aromatic amino acids also play important roles in plant defense and development.In bacteria, fungi and plants, the three aromatic amino acids, Trp, Phe and Tyr, are synthesized from a common precursor, chorismate that originates from the shikimate pathway ( Figure 1). In bacteria, this pathway is almost exclusively used to produce aromatic amino acids for protein synthesis but, in higher plants, this pathway leads to the production of numerous aromatic secondary metabolites. The importance of this pathway in plants is indicated by the fact that about 20% of the carbon fixed by photosynthesis flow through this pathway. These facts strongly suggest that the modification of the shikimate and its derivative (or downstream) pathways may lead to dramatic changes in secondary metabolism. The enzymatic reactions in the biosynthetic pathway for aromatic amino acids seem to be identical in prokaryotes and eukaryotes but they apparently differ in the regulatory mechanisms for these pathways. For example, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the enzyme that catalyzes the first reaction in the shikimate pathway, is not feedback inhibited by any of the aromatic amino acids in plants, but, each of the three DAHP synthase isoenzymes are Abstract Aromatic amino acids function as building blocks of proteins and as precursors for secondary metabolism. To obtain plants that accumulate tryptophan (Trp) and phenylalanine (Phe), we modified the biosynthetic pathways for these amino acids in rice and dicot species. By introducing a gene encoding a feedback-insensitive anthranilate synthase (AS) alpha subunit, we successfully obtained transgenic plants that over-accumulated Trp. In addition, we found mutant calli that accumulated Phe and Trp at high concentrations. The causal gene (mtr1-D) encoded an arogenate dehydratase (ADT)/prephenate dehydratase (PDT) that catalyzes the final reaction in Phe biosynthesis. The wild-type enzyme was sensitive to feedback inhibition by Phe, but the mutant enzyme encoded by mtr1-D was relatively insensitive. Further, ...

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“…Although Arabidopsis tha/iana cDNAs which can complement trpE (Niyogi and Fink, 1992) and trpG (Niyogi eta/., 1993) mutants of E. coil have been described, the structure and biochemistry of the plant enzymes themselves are still obscure. Studies with crude extracts from corn and pea (Hankins et aL, 1976) as well as with the partially purified enzyme from Catharanthus roseus (Poulsen et aL, 1993) have led to speculation that AS in these species possesses a monofunctional J3-subunit. But AS from plants is difficult to purify and, as a consequence, no direct evidence has yet been marshalled which conclusively demonstrates the function and size of AS~ in plants.…”

Section: Introductionmentioning

confidence: 99%

Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

et al. 1995

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SummaryRuta graveolens utilizes anthrenilete synthese (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670-and 1700-fold, respectively. Glutamine-end ammoniadependent AS activities were strictly co-purified in all steps. Through cDNA cloning and complementetion of Escherichia coli deletion mutants defective for AS, it is shown that young Ruta plants express two genes for functional AS~ subunits, AS~ land AS(~2. The data indicate that AS~ from Ruta requires an AS~ subunit with a native molecular weight of 60-65 kDe for the glutaminedependent reaction. Protein synthesized in vitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature AS~ subunits are active in plastids in vivo. AS~I and AS(~2 are constitutively expressed in Ruta cell cultures, but AS~I steedy-stete mRNA levels are increased 100-fold 6 h subsequent to elicitation whereas AS~2 expression remains constitutive. Increased AS~I transcription corresponds to elicitorinduced alkaloid accumulation. The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.

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Purification and characterization of anthranilate synthase from Catharanthus roseus

Cited by 73 publications

References 36 publications

Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice

Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

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