Roles of chorismate mutase, isochorismate synthase and anthranilate synthase in plants

Poulsen, Charlotte; Verpoorte, Robert

doi:10.1016/0031-9422(91)83688-h

Cited by 78 publications

(29 citation statements)

References 53 publications

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“…The flow was 0.5 mVmin and fractions of 0.5 ml were collected. AS activity peak fractions from Mono Q were applied at 200 pV run to a Superose 6 HR 10/30 column equilibrated with Superose 6 buffer (20 mM triethanolamine pH 7.5, 10% glycerol, 10 mM L-glutamine, 1 mM dithiothreitol). A summary of the purification is provided in Table 1.…”

Section: Purification Of Anthranilate Synthase Activitymentioning

confidence: 99%

Purification and characterization of anthranilate synthase from Catharanthus roseus

Poulsen¹,

Bongaerts²,

Verpoorte³

1993

European Journal of Biochemistry

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Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethy1ene glycol) precipitatiodfractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 f 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 f 5 kDa. The enzyme had a PI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH,' as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg", K,,, values for L-glutamine was determined to be 0.37f0.05 mM, for chorismate 67?3 pM, and for MgCl, 0.26 -C-0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperativity of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20pM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperativity for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.Anthranilate synthase (AS; EC 4.1.3.27) catalyzes the first reaction branching from the shikimate pathway toward the biosynthesis of tryptophan occurring in bacteria, fungi and plants [I] (Fig. 1).Tryptophan is a key precursor for the indole alkaloids, among which there are several pharmacologically important compounds [2], from Catharanthus roseus, for example, the two anticancer drugs vinblastine and vincristine. The availability of tryptophan might be a limiting factor in the production of indole alkaloids. In all plants and cell cultures where AS has been examined, the enzyme was found to be strongly feedback-inhibited by tryptophan ; in accordance with this, several studies have indicated that loss of feedback regulation of AS can lead to largely unregulated accumulation of tryptophan [3 -61.AS enzymes have been purified to homogeneity and characterized from a number of microorganisms. In all mi- crobial species studied, AS is composed of two nonidentical subunits [7-91. Component I (also called a-subunit) binds the substrate chorismate and catalyzes its aromatization; component I1 @-subunit) binds the other substrate glutamine and transfers an ammonia group from glutamine to component I (chorismate). This glutamine-dependent AS reaction requires both subunit...

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Section: Purification Of Anthranilate Synthase Activitymentioning

confidence: 99%

Purification and characterization of anthranilate synthase from Catharanthus roseus

Poulsen¹,

Bongaerts²,

Verpoorte³

1993

European Journal of Biochemistry

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“…2,3) In plants, tryptophan biosynthesis is strictly controlled; anthranilate synthase, the first committed enzyme, is feedback-inhibited by micromolar concentrations of tryptophan, resulting in low levels of soluble tryptophan. 4) Two recent studies found that feedback inhibition of tryptophan biosynthesis via anthranilate synthase is disarmed when plants are subjected to pathogen infection or senescence. 5,6) For example, upon senescence, rice leaves begin to accumulate tryptophan at up to 1.6 mg per g fresh weight (fw) in parallel with the induction of anthranilate synthase transcripts.…”

mentioning

confidence: 99%

Tryptophan Boost Caused by Senescence Occurred Independently of Cytoplasmic Glutamine Synthetase

Lee

Kang

Kim

et al. 2010

Bioscience, Biotechnology, and Biochemistry

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“…Low levels of AS activity have been reported in the crude extract or after one purification step from several plant cultures as well as whole plant sources (Poulsen and Verpoorte, 1991). AS from plants has not been purified to homogeneity.…”

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confidence: 99%

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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Roles of chorismate mutase, isochorismate synthase and anthranilate synthase in plants

Cited by 78 publications

References 53 publications

Purification and characterization of anthranilate synthase from Catharanthus roseus

Purification and characterization of anthranilate synthase from Catharanthus roseus

Tryptophan Boost Caused by Senescence Occurred Independently of Cytoplasmic Glutamine Synthetase

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

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