EPSP, 5-enolpyruvylshikimate 3-phosphat

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

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). 

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

The concept is not limited to analogs of simple linear organophosphates (e.g. phosphoenol pyruvate analog as inhibitor of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase [71]) but can be extended to cyclic phosphates (e.g. cyclic phosphoinositol analog as a phospholipase C inhibitor [72]) and transition state analogs (e. g. purine nucleoside phosphorylase (PNP) inhibitor [73]) (Scheme 4.30). 

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). 

EPSPS a single copy of the gene coding for glyphosate tolerance CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) bom Agrobacterium sp. strain CP4. 

The conversion of shikimate-3-phosphate (7) to 5-enolp-yruvylshikimate (EPSP) (8) by 5-enolpyruvylshikimate-3-phosphate synthase represents a rare type of reaction in which the enolpyruvate fragment of phosphoenolpyruvate (2) is transferred to a molecule of (— )-shikimate-3-phos-phate (7) (Figure 7.1). The herbicide glyphosate (10) blocks the enzyme that catalyzed this reaction (Fig. 7.3). In 1984, sales of this herbicide totalled 480 million (Amrhein, 1986 Floss, 1986). 

By means of phosphoenolpyruvate labeled with both tritium and deuterium ( -[3- Hi, Hi]phosphoenolpyruvate), it is possible to determine the stereochemistry of both the addition and the elimination step involved in the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) (8) (Floss, 1986) (Fig. 7.3). The EPSP synthase reaction involves addition and elimination at the 2,3 double bond with opposite stereochemistry (i.e., either syn addition and anti elimination or anti addition followed by syn elimination) (Floss, 1986). 

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. 

Enolpyruvylshikimate-3-phosphate (EPSP) synthase is where the money lies. Its identification by Nikolas Amrhein and Hans Steinriicken as the site of action of the blockbuster herbicide glyphosate awakened commercial interest in the shikimate path-... 

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/ ... 

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. 

Enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), also known as 3-phosphoshikimate 1-carboxyvinyltransferase, catalyzes the reversible transfer of the enolpyruvyl group from PEP to shikimate 3-phosphate (Figure 2.2). There is no known cofactor requirement. When purified to homogeneity, bacterial and plant EPSP synthases are monomeric monofunctional proteins of molecular weight 40,000-50,000. " Some fungi have a radically different molecular architecture in which EPSP synthase is part of a pentafunctional polypeptide which also contains the previous four enzymatic activities of the common shikimate pathway, the arom complex. 

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