Enolpyruvylshikimic acid phosphate

The effects of glyphosate on phenolic compound production are two-fold 1) accumulation of phenolic compounds that are derivatives of aromatic amino acids is reduced and 2) pools of phenolic compounds derived from constituents of the shikimate pathway prior to 5-enolpyruvylshikimate-3-phosphate become larger. Assays that do not distinguish between effects on these two groups, such as that for hydroxyphenolics of Singleton and Rossi (18), can lead to equivocal and difficult to interpret results (e.g. 3-5). 

Jaworski (4) reported that growth inhibition of both plant and microbes by glyphosate could be reversed by aromatic amino acids. Further work of Amrhein and his coworkers revealed that glyphosate inhibits the shikimate pathway enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (5). This enzyme catalyzes the reaction shown in Figure 1. Glyphosate-treated plant and bacterial cultures accumulate shikimate and/or shikimate 3-phosphate (S3P), confirming that inhibition of EPSPS is at least a part of the in vivo mechanism of action of this herbicide (6, 7). 

For example, glyphosate inhibits the enzyme, EPSP (5-enolpyruvylshikimate 3-phosphate) synthase, that catalyzes a step in the synthesis of the aromatic amino acids. Similarly, both the imidazolinones and sulfonylureas inhibit acetolactate synthase (ALS), the enzyme that catalyzes the first step in the formation of branched-chain amino acids (11). Triazine herbicides act by binding to a specific protein in the thylakoid membranes of the chloroplasts, preventing the flow of electrons and inhibiting photosynthesis (12). 

Plants synthesize all 20 common amino acids de novo. Glyphosate, a weed killer sold under the trade name Roundup, is an analog of phosphoenolpyruvate that specifically inhibits 3-enolpyruvylshikimate 5-phosphate synthase, a key enzyme of the pathway for chorismate biosynthesis. This compound is a very effective plant herbicide, but has virtually no effect on mammals. Why ... 

A 1,4-conjugate elimination of phosphoric acid then transforms 5-enolpyruvylshikimate-3-phosphate (8) into chorismate (9) (Fig. 7.1). The 6-pro-R hydrogen atom is labile in the chorismate synthetase reaction hence, the reaction occurs with overall trans-g ometry. 

Phosphoenblpyruvic acid (D 2) and erythrose-4-phosphate serve as precursors. 3-Deoxy-D-arabinoheptulosonic acid-7-phosphate is built as a key intermediate. This compound cyclizes to 5-dehydroquinic acid, which is transformed to 5-de-hydroshikimic acid and shikimic acid. After phosphorylation shikimic acid reacts with phosphoenolpyruvate. The formed 3-enolpyruvylshikimic acid-5-phosphate yields chorismic acid by an anti-elimination of a proton and the phosphate group. 

Deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase 2 5-dehydroquinate synthase 3 quinate dehydrogenase 4 5-dehydroquinate dehydratase 5 shikimate dehydrogenase 6 shikimate kinase 7 3-enolpyruvylshikimate-5-phosphate synthase 8 chorismate synthase... 

When the entire chain of seven common intermediates had been elucidated, the surprising fact emerged that this sequence oscillates back and forth between phosphorylated and phosphorus-free stages. It begins with 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), continues with the three phosphorus-free metabolites 3-dehydroquinic acid, 3-dehydroshikimic acid, and shikimic acid, proceeds next through the two phosphorylated intermediates shikimic acid 3-phosphate and 5-enolpyruvylshikimic acid 3-phosphate, and ends with the phosphorus-free chorismic acid. 

Research in David Sprinson s laboratory at Columbia University, in part contemporary with the research outlined in the preceding pages, has unraveled the earliest stages of the sequence and the derivation of the first, non-cyclic intermediate, DAMP. In the same laboratory, Judith Levin discovered 5-enolpyruvylshikimate 3-phosphate and predicted the existence and correct structure of the next, and last one, of the common intermediates, the compound from which the pathways to the individual primary aromatic products branch off. Isolation and structure proof of this branchpoint intermediate, chorismic acid, by the Australian workers F. Gibson, L.M. Jackman, and J.M. Edwards completed the elucidation of the general pathway. 

In this chapter, the discussion will concentrate on two inhibitors with a reasonable claim to selective action on enz3ones related to the shikimate pathway glyphosate, which inhibits 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase and L-a-aminooxy-3 phenylpropionic acid (L-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL) (Fig. 2). In addition to introducing a novel inhibitor of PAL, (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP), previous and current efforts to design inhibitors of other shikimate pathway enzymes will be described. The treatment presented here will show that the deductions and predictions made on the basis of the abstract scheme in Figure 1 can be, and have been, tested on the basis of the real pathway presented in Figure 2. 

The information obtained from the application of glyphosate to complex systems strongly pointed to one of the following three enzymes as the target of the inhibitor in the shikimate pathway shikimate kinase (EC 2.7.1.71), 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19), and chorismate synthase (EC 4.6.1.4). Jointly, these three enzymes convert shikimic acid to chorismic acid in a series of interesting reactions >(Fig. 2). A defined system" had therefore to be found in which the conversion of shikimic acid to chorismic acid could be conveniently studied. 

When we began our synthetic program, shikimic acid had been prepared by a number of routes, and the groups of Danishefsky and Plieninger had reported syntheses of prephenic acid. The intervening intermediates, shikimate-3-phosphate (S-3-P), 5-enolpyruvylshikimate-3-phosphate (EPSP), and chorismate, were only available from natural sources or by enz)onatic transformations. 

A 1,4-conjugate elimination of phosphoric acid transforms 5-enolpyruvylshikimate-3-phosphate (13) to chorismate (14). Two... 

The early studies concerning this compound centred on its dephosphorylated form 5-enolpyruvylshikimic acid (48) which was shown to accumulate, in addition to (—)-shikimic acid and shikimic acid-3-phosphate, in the media of several multiple aromatic bacterial auxotrophs blocked beyond shikimate . The compound... 

Enolpyruvylshikimic acid-3-phosphate (49) is best prepared enzymically from shikimic aci(i-3-phosphate and phosphoenolpyruvate using an ammonium sulpWe fraction from Escherichia coli K-12 mutant 58-278 and is normally isolated as its barium salt. Its proton magnetic resonance spectrum (potassium salt in deuterium oxide) has been reported by Sprinson and his collaborators and analysis of the various coupling constants indicated that... 

The synthesis of compound (44) as a potential transition-state analogue inhibitor of isochorismate synthase (IS) has been reported/ Compounds (45) and (46) have been synthesized from the known 6-fluoroshikimic acids (J. Chem. Soc., Chem. Commun., 1989, 1386) by treatment first with shikimate kinase then 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and shown to be competitive inhibitors of chorismate synthase/ ... 

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). 

The study of inhibitors of photosynthetic carbon metabolism has, by contrast, been remarkably unsuccessful. Attempts to design herbicidal inhibitors on rational grounds on the basis of specific enzyme inhibition have not afforded herbicidal compounds that are effective on whole plants. It is possible that in many instances the chloroplast envelope proves to be an insuperable barrier. Nevertheless, recent work identifying specific enzymes such as 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, acetyl-CoA carboxylase, and acetohydroxy acid synthase has demonstrated the effectiveness of enzyme inhibitors, although none were developed on rational grounds. 

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