Inhibition of glutamine synthase

The ammonium salt of glufosinate (DL-homoalanin-4-yl [methyl] phosphonic acid) is a major non-selective, post-emergence herbicide introduced in 1981 for the rapid control of vegetation. It is a potent inhibitor of glutamine synthase and, in common with methionine sulfoximine and tabtoxinine-P -lactam, is a structural analogue of glutamic acid (Figure 9.2).

The discovery and development of glufosinate are of particular interest since this herbicide may be regarded as having a natural origin. Glufosinate, also termed phosphi-nothricin, and a tripeptide containing glufosinate bound to two molecules of L- alanine (phosphinothricyl-alanyl-alanine) were first described in 1972 as products of the soil bacterium Streptomyces viridichromogenes. Independent studies in Japan in 1973 also discovered this tripeptide in culture filtrates of another soil bacterium, Streptomyces hygroscopicus, and this compound was introduced commercially as bialaphos in the 1980s. Bialaphos itself is inactive, but it is rapidly hydrolysed in plants to form the phy-totoxic glufosinate (Wild and Ziegler, 1989). An additional tripeptide, phosalacine, also produced by a Streptomyces sp. has been reported by Omura et al. (1984). This tripeptide differs from bialaphos in that one alanine moiety is replaced by leucine. Evidence for the inhibition of plant glutamine synthase by glufosinate was first provided by Leason and colleagues (1982) who demonstrated a Ki value of 0.073 mM, and this potent inhibition has since been confirmed in a wide variety of plants and algae.

Glutamine synthase (E.C. 6.3.1.2) catalyses the conversion of L-glutamate to L-glutamine in the presence of ATP and ammonia. This enzyme has a molecular weight of about 400 kDa and has eight subunits, each with an active site. It exists as separate isozymes in the leaf cytoplasm, chloroplast and roots, and in legumes as an isoform specific to root

Figure 9.1 Amino acid biosynthesis. R5P, ribulose 5-phosphate; PEP, phosphoenoyipyruvate; E4P, erythrose 4-phosphate; RuBP, ribuiose 1,5-bisphosphate; PGA, 3-phosphogiyceric acid.

CH2 NH

Glutamate Glufosinate Methionine Tabtoxinine-

sulfoximine p-lactam

Figure 9.2 The structures of glutamate and the glutamine synthases inhibitors glufosinate, methionine sulfoximine and tabtoxinine-p-lactam.

enzyme + L-glutamate

ATP ADP

Mg2+

enzyme y-glutamyl phosphate complex

L-glutamine

enzyme

Figure 9.3 The two step process in the catalysis of glutamine synthesis by glutamine synthase.

nodules. The isoform found in the chloroplast is expressed in greater amounts in the presence of light and at high sucrose concentrations, an indicator of high levels of photosyn-thetic activity. Glutamine synthase forms glutamine in a two-step process (Figure 9.3).

Normally, y-glutamyl phosphate is first produced from ATP and L-glutamate, and then ammonia reacts with this complex to release Pi and L-glutamine. However, glufosinate, as an L-glutamate analogue, can also be phosphorylated to produce an enzyme-glufosi-nate-phosphate complex to which ammonia cannot bind and the enzyme is irreversibly inhibited (Manderscheid and Wild, 1986) . Glufosinate can inhibit bacterial, plant and mammalian glutamine synthase in vitro, but is non-toxic to mammals, apparently because of its inability to cross the blood-brain barrier and its rapid clearance by the kidneys (Kishore and Shah, 1988).

As with other herbicides that inhibit amino acid biosynthesis, the sequence of events leading from glufosinate application to plant death has been open to much debate. Phytotoxicity is certainly rapid, since leaf chlorosis, desiccation and necrosis may be observed within two days after treatment, and plant death results three days later. Since glutamine synthase is potently inhibited, the rapid accumulation of ammonia was the presumed toxic species which was thought to uncouple electron flow from proton transport in the thylakoid, so that photosynthesis was rapidly inhibited. This view was supported experimentally by the observation that phytotoxic symptoms only develop in plants exposed to light, especially when photorespiration is favoured. Thus, the ammonia generated in photorespiration is not reassimilated and may directly uncouple proton gradients.

Further and more recent observations clearly imply an additional and very rapid action of glufosinate on membrane transport processes that precede visual symptom development. Thus, treated plants accumulate ammonia and show increased rates of cell leakage of potassium ions, and the uptake of nitrate and phosphate are adversely affected. Indeed, Ullrich and colleagues (1990) have convincingly demonstrated that glufosinate, like glutamate, can directly cause an electrical depolarisation of the plasmalemma, although unlike glutamate, recovery is often incomplete and a secondary depolarisation is evident. These decreased membrane potentials inhibit or alter transport processes so that, for example, the cotransport of glutamate/H+ was irreversibly inhibited but potassium flux was increased. In this way, the accumulation of ammonia may be seen to uncouple or interfere with membrane function and transport at the plasmalemma as well as at the thylakoid.

Increases in ammonia concentrations in treated tissues have been reported as 10 times higher than in untreated tissues 4 hours after treatment and as high as 100 times within 1 day. Free ammonia is toxic to biological systems and it is for this reason that a number of processes are found in biological systems that aim to maintain free ammonia at very low concentrations.

An associated decrease in the concentration of a number of amino acids is also noted where treated plants remain in the light. A decrease in photosynthetic carbon fixation is also noted under the same conditions. These symptoms develop far slower in the dark and this is mirrored in field observations that glufosinate activity is observed far quicker under conditions of full sunlight.

Free ammonia has a severe affect on the pH gradient across biological membranes, collapsing membrane potentials. Glufosinate does not have a direct inhibitory effect on photosynthetic carbon fixation but was assumed to inhibit this process via the reduction in ATP production by photophosphorylation, due to the effect of ammonia on membrane potentials. However, Wild et al. (1987) reported that under conditions in which photorespiration was not taking place, photosynthesis was not inhibited by the increased ammonia concentrations resulting from glufosinate treatment. Photorespiration is an alternative metabolic pathway where ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) - the enzyme responsible for carbon fixation in the Calvin cycle using the products of the light stage of photosynthesis - uses oxygen in place of carbon dioxide as a substrate to react with ribulose 1,5-bisphosphate. Photorespiration takes place in conditions of elevated oxygen and, although it does produce glyceraldehyde 3-phosphate for the carbon reduction cycle, it does this at an energetically far less economical rate compared with the carboxy-lase activity of RuBisCo. In addition, photorespiration produces free ammonia that must be detoxified by reassimilation into organic molecules. Glufosinate will inhibit this reas-

similation and will therefore reduce or inhibit photorespiration, due to reduced concentrations of glutamate as an amino donor for glyoxylate. This will result in a reduction in photosynthetic carbon fixation, due to increased glyoxylate concentrations, and may also result in an increase in active oxygen species from triplet state chlorophyll in the light stage of photosynthesis. The result of this would be lipid peroxidation as reported for a number of other herbicide classes (Chapter 5).

It therefore appears that glufosinate can affect a number of metabolic pathways in plants by its indirect affect on membrane polarisation, reduced peptide, protein and nucleotide concentration, increased protein degradation (to release free amino acids) and the inhibition of photosynthetic carbon assimilation via the inhibition of photorespiration.

Introduction of genes encoding a specific glufosinate-metabolising enzyme has allowed the successful development of glufosinate-resistant, genetically modified crops. These are discussed in detail in Chapter 13.

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