Anthranilic acid chorismate synthase

Chorismic acid (26), thus, represents the first divergence point of pyocyanin from other biosynthetic pathways. The first authentic pyocyanin biosynthetic enzyme is PhzE, which has sequence similarity to anthranilate synthases, which generate anthranilate from chorismate. PhzE is thought to catalyze the conversion of chorismic acid (26) to amine 165. Compound 165 is in turn a substrate for PhzD, an isochorismatase that catalyzes the hydrolysis of the vinyl ether to 166 and pyruvate [188, 189],... 

For the aromatic pathway (Figure 30.20), the critical control points are the condensation of phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate 7-phosphate, DAHP, by DAHP synthase. For tryptophan, the formation of anthranilic acid from chorismic acid by anthranilate synthase is the second critical control point. The transcriptional regulation was overcome through the use of alternative promoters and allosteric regulation was circumvented by the classical technique of selection for feedback-resistant mutants using toxic analogues of the repressing compounds. 

Biosynthesis Like other aromatic amino acids, e.g., Phe and Tyr, Trp is formed on the shikimic acid pathway. There is a branching point at chorismic acid one branch leads to Phe and Tyr, the other to Trp choris-mic acid - anthranilic acid (anthranilic acid synthase, EC 4.1.3.27)- A-(5 -0-phosphoribosyl)-anthranilic acid (anthranilic acid phosphoribosyl transferase, EC 2.4.2.18)- 1 -o-carboxyphenylamino-1 -deoxyribu-lose 5-phosphate [A-(5 -phosphoribosyl)anthranilic acid isomerase]- indole-3-glycerol phosphate (in-dole-3-glycerol phosphate synthase, EC 4.1.1.48) - indole (tryptophan synthase, EC 4.2.1,20)+serine - Trp. Many biologically active indole compounds are derived from Trp, e. g., 5-hydroxytryptophan, 5-hydroxy-tryptamine ( serotonin), and melatonin as well as many indole alkaloids. 

The first step in the formation of tryptophan involves conversion of chorismate (9) to anthranilate (11) (Fig. 7.4). Although the reaction is not well understood, it is catalyzed by the enzyme anthranilate synthase and utilizes L-gluta-mine. By means of specifically labeled chorismic acid, it was determined that the protonation involved in the formation of anthranilic acid had occurred from the re face (Figure 4) (Floss, 1986). Anthranilic acid (11) also serves as an intermediate for the synthesis of a number of secondary compounds and occurs free and as various derivatives in many plants and other organisms (Dewick, 1989). 

Anthranilic acid (or o-amino-benzoic acid) is an aromatic acid with the formula C H NO, which consists of a substituted benzene ring with two adjacent, or "ortho- functional groups, a carboxylic acid, and an amine (Fig. 14.1). Anthranilic acid is biosynthesized from shikimic acid (for shikimic acid biosynthesis, see Chapter 10) following the chorismic acid-mediated pathway [1]. Based on its biosynthetic mechanism, shikimate is transformed to shikimate 3-phosphate with the consumption of one molecule of ATP, catalyzed by shikimate kinase. 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase is then catalyze the addition of phosphoenolpyruvate to 3-phospho-shikimate followed by the elimination of phosphate, which leads to EPSP. EPSP is further transformed into chorismate by chorismate synthase. Chorismate reacts with glutamine to afford the final product anthranilate and glutamate pyruvate catalyzed by anthranilate synthase (Fig. 14.1). 

Isochorismate synthase, that might be involved in the biosynthesis of 2,3-DHBA, has recently been purified from Catharanthus roseus cell-suspension cultures and subsequently its gene was cloned (L. van Tegelen, P. Moreno, A. Croes, G. Wullems and R. Verpoorte, submitted for publication). Two isoforms of the enzyme were purified and characterized. Both have an apparent molecular mass of 65 kD. The Km values for chorismic acid are 558 pM and 319 p.M for isoform I and II respectively. The enzymes are not inhibited by aromatic amino acids and require Mg for enzyme activity. The isolated cDNA encodes a protein of 64 kD with a A-terminal chloroplast targeting signal. The deduced amino acid sequence shares homology with bacterial isochorismate synthases, and also with anthranilate synthases, another chorismate utilizing enzyme. 

The pivotal position occupied by chorismic acid in the shikimic acid pathway has been established in several higher plants as well as microorganisms (Fig. 2) (Edwards and Jackman, 1965 Cotton and Gibson, 1968 Schmit and Zalkin, 1969 Gilchrist et al., 1972). By action of chorismate mutase [Fig. 3 (8)], chorismate is converted to prephenate which is subsequently metabolized by two independent pathways [Fig. 3 (9 and 11)] to form phenylalanine and tyrosine. Alternatively, chorismate serves as a substrate for anthranilate synthase, the first enzyme in the pathway branch leading to the synthesis of tryptophan [Fig. 4 (13)]. 

Fig. 6. Regulation of the synthesis of aromatic amino acids in plants. Each of the sequential arrows represents the enzyme catalyzed steps detected in Figs. 2, 3 and 4. Bold lines indicate inhibition of chorismate mutase by phenylalanine and tyrosine in addition to anthranilate synthase inhibition by tryptophan. The dashed arrow symbolizes the ability of tryptophan to both activate chorismate mutase and antagonize inhibition of this enzyme by either phenylalanine or tyrosine.

Figure 5.41 Early steps of the proposed indole acetic acid biosynthesis pathways for Ara-bidopsis. CHO, chorismate ANA, anthranilate PANA, 5-phosphoribosylanthranilate CADP, l-(o-carboxyphenylamino)-l-deoxyribulose-5-phosphate IGP, indole-3-glycerol phosphate TRP, tryptophan. Enzymes ASA, anthranilate synthetase, suhunit a ASB, anthranilate synthetase, suhunit P PAT, phosphorihosylanthranUate transferase PAI, phosphoiibosylanthrani-late isomerase IGS, indole-3-glycerol-phosphate synthase TSA, tryptophan synthase, subunit a and TSB, tryptophan synthase, suhunit p.

Fig. 145. Transformation of chorismic acid to hydroxy and aminobenzoic acids 1 Isochorismate synthase 2 anthranilate synthase...

Having chorismic acid available with a stereospecific label in the side chain methylene group we then proceeded to determine the steric course of the anthranilate synthase reaction. The pyruvate generated in the reaction in the presence of H2O (Fig. 19) was oxidized to acetate and analyzed for its chirality. An F value of 44.5 (19% e.e. indicated that the protonation had occurred on the face. This stereochemistry is probably of no mechanistic significance, but it contrasts with that seen in virtually all the reactions in which a proton or other electrophile is added at the methylene carbon of PEP. 

Fig. 9. Sequential pattern of allosteric control over biosynthesis of aromatic amino acids in the plastid compartment. In the presence of excess aromatic amino acids, L-tyrosine (TYR) inhibits arogenate dehydrogenase, L-phenylalanine (PHE) inhibits arogenate dehydratase and L-tryptophan (TRP) inhibits anthranilate synthase. The three aromatic amino acids exert allosteric inhibition (-) or activation (+) effects upon chorismate mutase-1 as symbolized. However, activation dominates over inhibition. The outcome of these events is to trap L-arogenate (AGN) between the various foci of control in the pathway. As shown symbolically, -arogenate (AGN) then acts to feedback inhibit DAHP synthase-Mn.

Scheme 11.85. A representation of the conversion of chorismate to anthranilate (under the control of anthranilate synthase, EC 4.1.3.27) and the conversion of chorismate to p-aminobenzoic acid (PABA), under the control of aminodeoxychorismate synthase (EC 2.6.1.85), to 4-amino4-deoxychorismate and thence to 4-aminobenzoic acid and pyruvate.The attractive common bicyclic lactone intermediate shown, although not required, is based on a suggestion of Walsh, C. T. Liu, J. Rusnak, E Sakaitani, M. Chem. Rev., 1990, 90,1105. EC numbers and some graphic materials provided in this scheme have been taken from appropriate links in a URL starting with http //www.chem.qmul.ac.uk/iubmb/enzyme/.

Figure 7. Metabolic pathways that use chorismate as a starting material The SgcD/SgcG-dependentpathway is specific to C-1027 biosynthesis (shaded) and represents the newest pathway originating from chorismate. ADC, 4-amino-4-deoxychorismate ASI, anthranilate synthase component I IC, isochorismate SA, salicylic acid ADIC, 2-amino-2-deoxyisochorismate OPA, 2-enolpyruvylanthranilate.

Of the enzymes associated with aromatic amino acid biosynthesis, there is good evidence that unique isozymes of DAHP (Rubin and Jensen, 1985), chorismate synthetase (d Amato et al 1984), and anthranilate synthase (Brotherton et ai, 1986) are differentially localized within chloroplasts and in the cytoplasm. The regulatory properties of the plastid isozymes are consistent with their involvement in amino acid synthesis. Many of the remaining pathway enzymes have also been detected in plastids, including all those required for the synthesis of EPSP [(1) to (6)] (Mousdale and Coggins, 1985). These results, combined with those obtained during measurements of the biosynthetic capabilities of isolated chloroplasts (Bickel and Schultz, 1979 Buchholz and Schultz, 1980 Schulze-Siebert et ai, 1984), leave little doubt that these organelles are a primary site of aromatic amino acid biosynthesis. 

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