Enolpyruvate shikimate-3-phosphate

Enolpyruvate shikimate-3-phosphate synthase (EPSPS) is the enzyme that catalyzes the condensation of shikimate-3-phosphate with phosphoenolpymvate. The corresponding difluorophosphonate (phosphoenolpymvate analogue) irreversibly inhibits EPSPS. The mechanism of the inhibition by difluorophosphonate is similar to that reported for MurZ inhibition (Figure 7.31). ... 

The conversion, by bacterial extracts, of D-oZtro-heptulose 1,7-diphosphate to shikimate, essentially without side reactions, greatly facilitated subsequent study of the intermediate steps in the synthesis. It was shown that the addition of iodoacetate or fluoride completely blocks this conversion. In the presence of iodoacetate, synthesis is restored by the addition of either D-glyceronic acid 3-phosphate or enolpyruvate phosphate. In the presence of fluoride, only enolpyruvate phosphate is able to restore shikimate synthesis. Neither D-fructose 1,6-diphosphate nor pyruvate reverses these inhibitions. These results suggested that the reactions of glycolysis, from triose phosphate to enolpyruvate phosphate (see Fig. 2), are involved in the conversion of D-oZfro-heptulose diphosphate to shikimate. The effect... 

Enolpyruvate phosphate and n-erythrose 4-phosphate are independent intermediates in metabolic pathways of n-glucose that are not directly concerned with aromatic biosynthesis. Their simultaneous requirement for shikimate formation therefore indicates the first specific, or branch-point,... 

The view that such a reaction is the initial one in the conversion of enolpyruvate phosphate plus D-erythrose 4-phosphate to shikimate, was supported by fractionation of the crude, bacterial extracts and by the chemical synthesis of 3-deoxy-D-ara6mo-heptulosonic acid 7-phosphate. The steps in... 

The other compound, called Zl, which was much more acid-labile, was hydrolyzed to equimolar amounts of pyruvate and shikimate, and was tentatively assigned the structure of shikimate 3- or 5-enolpyruvate ether. In a more recent study, it was found that the barium salt of Zl does not absorb in the carbonyl region of the infrared absorption spectrum (no ester structure), and that it has a strong band at 8.2iu characteristic of a vinyl ether. It is oxidized very rapidly by periodate, giving rise to an unstable compound with maximum absorption at 235 m i ( = 4000). A similar unstable chromophore, most likely having the structure XVII, was produced by periodate oxidation of shikimate 3-phosphate but not of shikimate 5-phosphate. (3-Methyl-crotonaldehyde shows Xm 235, t = 6700. ) These observations suggest that Zl is shikimate 3-enolpyruvate ether (XVIII). 

Further study of this synthesis in partly purified extracts did not reveal any requirements for cofactor or metal. Shikimate 5-phosphate (alone) was extensively hydrolyzed in these extracts to shikimate and orthophosphate, but, when equimolar amounts of enolpyruvate phosphate were also added, only insignificant quantities of shikimate were produced, and, for every mole of Zl formed, two equivalents of orthophosphate were released. The phosphatase action on shikimate 5-phosphate was strongly inhibited when the amount of enolpyruvate phosphate present was equivalent to only 10 per cent of the shikimate 5-phosphate, but Zl added initially to shikimate 5-phosphate did not inhibit the phosphatase activity. 

Since shikimate 5-phosphate is a required precursor, and a 3-0-substi-tuted shikimate is the end product, it is suggested that the first intermediate in the reaction between shikimate 5-phosphate and enolpyruvate phosphate is the phosphorylated Zl (XIX) the latter is then hydrolyzed to Zl. Some evidence that this is, indeed, the case was obtained by the addi-... 

Structural considerations and known enzymic reactions suggest that the reaction of shikimate 5-phosphate with enolpyruvate phosphate is related to prephenate formation, and that the reactions of shikimate 5-phosphate with glutamine are involved in the synthesis of anthranilate and p-amino-benzoate. A common intermediate for these two branches of the main pathway, after shikimate 5-phosphate, appears to be unlikely, since it would require the further transformation of the latter compound before reaction with enolpyruvate phosphate. If shikimate 5-phosphate is the branch-point intermediate, quintuple auxotrophs should be completely blocked in any one reaction before shikimate 5-phosphate. In the absence of secondary metabolic effects, mutants blocked immediately after it should not be quintuple auxotrophs. They should show a requirement for either phenylalanine plus tyrosine, or tryptophan plus p-aminobenzoate (since anthranilate and p-aminobenzoate may have a common intermediate ). p-Hydroxybenzoate might be derived from this intermediate, or, independently, from shikimate 5-phosphate. This possibility is illustrated on page 264, X being the possible common intermediate. 

In the presence of partially purified extracts of E. coli, shikimate 5-phosphate and enolpyruvate phosphate react to yield 3-enolpyruvylshikimate 5-phosphate and orthophosphate (Levin and Sprinson, 1964). 3-Enolpyruvylshikimate 5-phosphate (ES-5-P) has been isolated as an essentially pure barium salt by anion exchange chromatography of incubation mixtures on Dowex 1 (Cl). The chemical properties of 3-enolpyruvylshikimate 5-phosphate and its infrared spectrum were consistent with the proposed structure (XCVII) ... 

Other challenges arise when using metabolites from different branches of metabolism. For example, the production of shikimates requires phospho-enolpyruvate, an intermediate of glycolysis, and erythrulose 4-phosphate, an intermediate. It is uncertain whether the fluxes of these would be properly balanced if the attempt were made to overexpress the shikimate pathway. In glucose medium, the production of DAHP was significantly improved by the expression of Pps as it increases the availability of PEP [30, 31,184]. 

Because shikimic acid does not enter into mammalian metabolism, its synthesis and use are clear targets at which to aim selective toxicity. In bacteria, shikimic acid arises by cyclization of the carbohydrate 3-deoxy-2-oxo-D- mAzVzoheptulosonic acid 7-phosphate, which is formed by the condensation of erythrose 4-phosphate and phosphoenolpyruvic acid. Shikimic acid undergoes biosynthesis to chorismic acid (4.55) which is the enolpyruvic ether of raw5-3,4-dihydroxy cyclohexa-1,5-diene-1-carboxylic acid. As its name indicates, this acid sits at a metabolic fork, the branches of which lead to prephenic acid, to phenylalanine (and hence to tyrosine), to anthranilic acid (and hence tryptophan), to ubiquinone, vitamin K, and/ -aminobenzoic acid (and hence folic acid). 

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). 

---