Inhibition of EPSP synthase

The herbicide glyphosate (N-phosphonomethyl glycine) (Figure 9.4) is a major non-selective, post-emergence herbicide used in circumstances where the total control of vegetation is required. Its success lies in very low soil residual activity, broad spectrum of activity, low non-target organism toxicity and great systemicity in plants, so that even the most troublesome rhizomatous weeds can be controlled. Monsanto received a patent for use of phosphoric acid derivatives, including glyphosate, as non-selective herbicides in 1974. The Stauffer Chemical Company, who had patented a number of phosphonic and phosphinic acids as industrial cleaning agents in 1964, subsequently released sulfosate (glyphosate-trimesium) for development in 1980.

The search for the target site of glyphosate action began with the observation by Jaworski (1972) that the control of duckweed (Lemna sp.) by glyphosate could be overcome by the addition of aromatic amino acids to the growth medium. However, it was not until 1980 that Steinrucken and Amrhein identified the enzyme 5 -enoyl-pyruvyl shikimic acid 3-phosphate (EPSP) synthase (E.C. 2.5.1.19) as being particularly sensitive to glyphosate. This enzyme is involved in the biosynthesis of the aromatic amino acids tryptophan, phenylalanine and tyrosine, and also leads to the synthesis of numerous secondary plant products (Figure 9.5). Approximately 20% of carbon fixed by green plants is routed through the shikimate pathway with an impressive number of significant end products, including vitamins, lignins, alkaloids and a wide array of phenolic compounds such as flavonoids. A common pathway exists from erythrose 4-phosphate, provided from photosynthetic carbon reduction in the chloroplast stroma, to chorismic acid, so inhibition at EPSP

Figure 9.4 The structure of glyphosate.

Phosphoenol pyruvate (PEP)

Erythrose 4-phosphate

COOH

shikimic acid H

H2CCNH2COOH

PHENYLALANINE

OH OH

shikimic acid 3 ^p^

H2CCNH2COOH

TYROSINE

TYROSINE

O COOH

COOH

OH OH

shikimic acid 3 ^p^

O COOH

chorismic acid

O COOH

chorismic acid

■ CH2CNH2COOH

TRYPTOPHAN

Figure 9.5 Biosynthesis of aromatic amino acids. EPSP, 5-enoylpyruvate shikimic acid 3-phosphate; *, EPSP synthase.

synthase by glyphosate is at a particularly strategic location. Additionally, the enzyme is not found in animals and so the chance of non-target organism toxicity is reduced.

The higher plant enzyme has a molecular weight of 45-50 kDa, found mainly in the chloroplast, which can be reversibly inhibited by glyphosate with an I- 0 of about 1020 |M. EPSP synthase is synthesised in the cytoplasm before being targeted to the chlo-roplast. For glyphosate to be active it must enter into the cell and then the chloroplast in sufficient concentration to inhibit the enzyme. Glyphosate is one of only a handful of herbicides for which a carrier protein has been identified that aids in the crossing of plasma membranes. In the case of glyphosate it is a phosphate carrier that is involved (Denis and Delrot, 1993 ).

Studies indicate that glyphosate acts as a competitive inhibitor with respect to phos-phoenolpyruvate (PEP, Ki; 0.1-10|M), but as a non-competitive inhibitor with respect to shikimic acid 3-phosphate (S3P). Mechanistically, S3P forms a complex with the enzyme to which glyphosate binds before PEP addition. Glyphosate appears to bind away from the active site of EPSP synthase, at a putative allosteric site, and also has very little affinity for the free enzyme. However, it is 'trapped' on the enzyme in the presence of EPSP. The binding of glyphosate appears to prevent the binding of PEP to the enzyme. Observations that even minor changes in the structure of glyphosate result in loss of binding and subsequent herbicidal activity suggest a very specific enzyme herbicide interaction.

A number of studies have attempted to use X-ray crystallography to resolve the order and binding characteristics of the substrates and glyphosate to EPSP synthase (Stilling et al., 1991; Franz et al., 1997; Schoenbrunn et al., 2001). Observations suggest that EPSP has two distinct hemispherical domains that come together in a 'screw-like' movement that causes the active site to be revealed. It appears that the binding of S3P is responsible for causing this conformational change. The active site, although yet to be identified, is postulated to be near the 'hinge' where the two domains of the EPSP synthase enzyme meet. A number of important, conserved, amino acids have been identified that appear to play important roles in active site binding and activity. The cleft formed between the two domains is largely electropositive and this plays a role in the attraction of the anionic ligands that are EPSP substrates (Figure 9.6).

Furthermore, since the enzyme is more active above pH 7, as may be expected in the chloroplast stroma in the light, it is the ionised form of glyphosate which is most likely to be the inhibitory species. Thus, S3P accumulates and the inhibition cannot be reversed by PEP.

Additional proof that EPSP synthase is the target enzyme for glyphosate has come from the elegant studies of Comai et al. (1985). These workers isolated a mutant form of EPSP synthase from Salmonella typhimurium which was resistant to glyphosate because of a single amino acid substitution, from proline to serine. Furthermore, they isolated the aroA gene encoding the resistant enzyme and successfully transferred it to tobacco, making this crop glyphosate resistant. Other studies have cloned the aroA gene and an over-production of EPSP synthase in plant cell cultures has also generated tolerance to glyphosate. Subsequent isolation of glyphosate-resistant EPSP synthase and incorporation into a number of crop species is discussed in detail in Chapter 13.

Glyphosate treatment causes growth inhibition, chlorosis, necrosis and subsequent plant death. Plants treated with glyphosate, however, may not show symptoms of treatment for 7-10 days. This slow action in the field probably reflects the time taken for

Figure 9.6 The X-ray crystal structure of E. coli EPSP synthase. On the left is the open form. On the right is the closed configuration. A modelled glyphosate molecule in the left form is shown, leading to the closed formation on the right (from CaJacob et al., 2004).

the depletion of aromatic amino acid pool sizes to cause decreased rates of protein synthesis. It is now known that glyphosate also has additional effects on phenol and pigment metabolism. Some studies have suggested that glyphosate induces a transient increase in the activity of phenylalanine ammonia lyase, an important enzyme in phe-nylpropanoid metabolism, so that enhanced concentrations of natural growth inhibitory phenols accumulate. This enzyme activity declines when phenylalanine pools become limiting. Since many phenols are regarded as natural inhibitors of auxin oxidation, then greater auxin metabolism occurs in glyphosate-treated plants with the result that apical dominance is overcome. Consequently, lateral growth of dicots and increased tillering in monocots are often observed in the field.

Relating EPSP synthase inhibition to plant death is not easy owing to the multiple effects this has on a number of metabolic processes. Inhibition of the enzyme results is reduced production of chorisimic acid and a subsequent increase in shikimate and shiki-mate-3-phosphate due to the blocking of the shikimic acid pathway. In glyphosate-treated tissue, shikimate and shikimatet 3tphosphate have been reported as making up 16% of plant dry matter (Schuktz et al., 1990). This causes a reduction in protein synthesis due to depletion of amino acid pools. This alone would result in growth retardation and subsequent plant death. However, blocking the shikimic acid pathway and subsequent reduction in chorisimate causes an increase in flow of carbon compounds out of the photosynthetic carbon reduction cycle. This results in reduction in both photosynthesis and starch production. Reduced carbon compound translocation and, ironically, glyphosate translocation from treated tissue are subsequently observed. In addition, blockage of the shikimic acid pathway results in reduction in the production of auxin growth regulators, lignin, plant defence compounds, UV protectants and photosynthetic pigments, either directly or via reduced synthesis of plastoquinone.

A further metabolic consequence of glyphosate treatment is the development of chlorotic areas on leaves. This may be due to an inhibition of 8-aminolaevulinic acid (8-ALA) synthetase, an early reaction in the biosynthesis of all porphyrin containing molecules, including the chlorophylls and cytochromes (Chapter 6, section 2). Since these compounds are central to plant metabolism, it would appear that many important biochemical pathways are therefore subject to interference by glyphosate treatment. Indeed, the observation that only 10 ||M glyphosate will inhibit EPSP synthase in vitro from field rates of greater than 10000|M, suggests the involvement of many secondary sites of action which produce an overall herbicidal effect.

Since glyphosate is a non-selective total herbicide, little is known about its metabolism in plants, which is presumed to be either non-existent or ineffectual. However, this herbicide is rapidly biodegraded by soil microorganisms which are able to utilise glyphosate as a sole phosphate source. Specific enzymes operate in a Pseudomonas sp. to cleave the phosphate group, and the phosphonomethyl C-N bond is broken to release glycine.

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