Aromatic Amino Acid Biosynthesis

Biochemical Interface Between Aromatic Amino Acid Biosynthesis and Secondary Metabolism... 

It is generally accepted that chloroplasts possess an intact pathway of aromatic amino acid biosynthesis that is tightly regulated. In addition, the subcellular location of some aromatic-pathway isozymes has been shown to be in the cytosol, but whether an intact pathway exists in the cytosol has not yet been proven. The evidence bearing on aromatic amino acid compartmentation and regulation is reviewed, with particular emphasis given to the relationship between primary biosynthesis and secondary metabolism in the cytosol. 

The tightly regulated pathway specifying aromatic amino acid biosynthesis within the plastid compartment implies maintenance of an amino acid pool to mediate regulation. Thus, we have concluded that loss to the cytoplasm of aromatic amino acids synthesized in the chloroplast compartment is unlikely (13). Yet a source of aromatic amino acids is needed in the cytosol to support protein synthesis. Furthermore, since the enzyme systems of the general phenylpropanoid pathway and its specialized branches of secondary metabolism are located in the cytosol (17), aromatic amino acids (especially L-phenylalanine) are also required in the cytosol as initial substrates for secondary metabolism. The simplest possibility would be that a second, complete pathway of aromatic amino acid biosynthesis exists in the cytosol. Ample precedent has been established for duplicate, major biochemical pathways (glycolysis and oxidative pentose phosphate cycle) of higher plants that are separated from one another in the plastid and cytosolic compartments (18). Evidence to support the hypothesis for a cytosolic pathway (1,13) and the various approaches underway to prove or disprove the dual-pathway hypothesis are summarized in this paper. 

JENSEN ETAL. Aromatic Amino Acid Biosynthesis Metabolism 99... 

Aromatic amino acids interface with a diverse and vast network of connecting secondary metabolism in the cytosol, but not in other major compartments such as the chloroplast. A strong rationale and emerging lines of experimental evidence support the probable existence of an intact cytosolic pathway of aromatic amino acid biosynthesis which links carbohydrate metabolism (via PEP and erythrose-4-P, or possibly glyceraldehyde-3-P) and secondary metabolism. 

Aromatic Amino Acid Biosynthesis. The shikimate pathway is the biosynthetic route to the aromatic amino acids tryptophan, tyrosine and phenylalanine as well as a large number of secondary metabolites such as flavonoids, anthocyanins, auxins and alkaloids. One enzyme in this pathway is 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) (Figure 2.9). 

In recent years, agribusiness firms have developed pf empirically several compounds that inhibit essential steps in the biosynthesis of amino acids found in plants but missing in animals. One of these compounds, glyphosate, is a highly specific inhibitor of 5-enol pyruvyl-shikimate-3-phosphate synthase (an enzyme needed for aromatic amino acid biosynthesis). Glyphosate is the active ingredient in the widely used herbicide Roundup. 

Aromatic amino acid biosynthesis proceeds via a long series of reactions, most of them concerned with the formation of the aromatic ring before branching into the specific routes to phenylalanine, tyrosine, and tryptophan. Chorismate, the common intermediate of the three aromatic amino acids, (see fig. 21.1) is derived in eight steps from erythrose-4-phosphate and phosphoenolpyruvate. We focus on the biosynthesis of tryptophan, which has been intensively studied by both geneticists and biochemists. 

Winkel-Shirley B. 1999b. Macromolecular organization of the primary and secondary pathways of aromatic amino acid biosynthesis. Physiol Plantarum 107 142-149. 

Glyphosate is toxic to plants and free-living microorganisms because it inhibits aromatic amino acid biosynthesis. On the other hand, it is extremely nontoxic to humans and animals because humans derive their amino acids from the diet. Additionally, it is broken down in the soil, so it is non-persistent. The only problem with glyphosate herbicides is that they will kill crop plants as readily as weeds. Recently, genetically engineered crop varieties have been introduced which are resistant to the herbicide, allowing weeds to be killed preferentially. 

The 4-aminobenzoate moiety of tetrahydrofolic acid is obtained from the shikimate pathway of aromatic amino acid biosynthesis via chorismate. Interestingly, apicomplexan protozoa may have conserved the complex shikimate pathway for the single purpose to generate 4-aminobenzoate as a tetrahy-drofolate precursor, whereas aromatic amino acids are obtained from external sources. 

Isotopic Evidence on the Pathways of Aromatic Amino Acid Biosynthesis... 

Overproduction of EPSPS has been observed in several plant cell cultures tolerant to glyphosate (12, 13, 29). In the case of glyphosate-tolerant Corydalis cultures, Smart et al. demonstrated by 2 D-gel electrophoresis, the overproduction of other proteins besides EPSPS. Since the levels of activity of several shikimate pathway enzymes were unaltered in the tolerant cell line compared to the parent cell line, it was concluded that these amplified proteins may not be involved in aromatic amino acid biosynthesis. It is possible that the other proteins may not have a role in the tolerance mechanism. Alterations in protein profiles between glyphosate-sensitive and tolerant petunia cell lines have also been observed. With the glyphosate tolerant carrot cell line, in addition to overproduction of EPSPS, the levels of aromatic amino acids were found to be enhanced (29). Based on the results with plant cell cultures, it was therefore not clear if overproduction of EPSPS was sufficient to obtain glyphosate tolerance in plants. 

Since aromatic amino acid biosynthesis occurs in the chloroplast of plants, and the petunia EPSPS was demonstrated to contain the information for its translocation to the chloroplast, it was of interest to determine if delivery of the bacterial mutant enzyme to chloroplast of transgenic plants affected the level of tolerance to Roundup . A hybrid gene was synthesized containing... 

It is clear from the above discussion ensure that glyphosate tolerance may be conferred to plants both by overproduction of wild type EPSPS as well as mutant EPSP synthases. It has been suggested that glyphosate may have multiple sites of action in plant cells (51-56). If this is true, mutant EPSPS enzymes would not confer glyphosate tolerance to plants, which is evidently not the case. It appears, therefore, that reports concerning the effect of glyphosate on other aspects of plant metabolism are due to secondary effects of the herbicide arising as a consequence of the inhibition of aromatic amino acid biosynthesis. 

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