Chorismate synthase aromatic amino acid biosynthesis

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. 

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. 

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. 

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.

Chorismate Synthase. - Chorismate synthase catalyses the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh and last enzyme of the shikimate pathway. Chorismate constitutes a major building block for the biosynthesis of an array of aromatic compounds, including the amino acids phenylalanine, tryptophan and tyrosine. Although this reaction does not involve a change in redox states, the enzyme requires reduced FMN for activity, and binds oxidized flavin only very weakly which results in its isolation as the flavin-free apo-enzyme. Macheroux and co-workers have used spectrophotometry, fluorimetry and EPR and ENDOR to investigate binding of the oxidized, reduced and radical forms of FMN to chorismate synthase in the presence of (6R)-6-fluoro-5-enolpyruvylshikimate-3-phosphate(a substrate ana-... 

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). 

---