Shikimate route, phenylalanine

Theoretically, many of the above discrepancies could be settled by experiments with carboxyl-labeled shikimic acid because this functional group would be lost in the formation of phenylalanine, but retained in the case of a direct conversion to gallic acid. Only ambiguous evidence was obtained, however, from such efforts (10), and it was concluded that at least two pathways for gallic acid biosynthesis must exist (14), with the preferential route depending on leaf age and plant species investigated (15,16). 

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). 

The biosynthesis of gallic acid (3.47) has been under investigation for more than 50 years. Different biosynthetic routes have been proposed, as depicted in Figure 3-6 (/) direct biosynthesis from an intermediate of the shikimate pathway, (2) biosynthesis via phenylalanine (3.27), cinnamic acid (3.29), />coumaric acid (3.30), caffeic acid (3.32), and 3,4, 5-trihydroxycinnamic acid (3.44), or (3) biosynthesis via caffeic acid (3.32) and protocatechuic acid (3.45). The possibility that different pathways co-existed in different species or even within one species was also considered. 

Fig. 4-2. Simplified reaction route illustrating the formation of lignin precursors. 1, 5-Dehydroquinic acid 2, shikimic acid 3, phenylpyruvic acid 4, phenylalanine 5, cinnamic acid 6, ferulic acid (Ri=H and R2=OCH3), sinapic acid (R,= R2=OCH3), and p-coumaric acid (R1=R2 = H) 7, coniferyl alcohol (Ri = H and R2=OCH3), sinapyl alcohol (Rj = R2=OCH3), and p-coumaryl alcohol (R =R2=H) 8, the corresponding glucosides of 7.

Aromatic compounds arise in several ways. The major route utilized by autotrophic organisms for synthesis of the aromatic amino acids, quinones, and tocopherols is the shikimate pathway. As outlined here, it starts with the glycolysis intermediate phosphoenolpyruvate (PEP) and erythrose 4-phosphate, a metabolite from the pentose phosphate pathway. Phenylalanine, tyrosine, and tryptophan are not only used for protein synthesis but are converted into a broad range of hormones, chromophores, alkaloids, and structural materials. In plants phenylalanine is deaminated to cinnamate which yields hundreds of secondary products. In another pathway ribose 5-phosphate is converted to pyrimidine and purine nucleotides and also to flavins, folates, molybdopterin, and many other pterin derivatives. 

Phenolic compounds include a wide range of secondary metabolites that are biosynthesised from carbohydrates through the shikimate pathway [14]. This is the biosynthetic route to the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, and only occurs in microorganisms and plants. In the first step, the glycolytic intermediate phosphoenol pyruvate and the pentose phosphate intermediate erythrose-4-phosphate are condensed to 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), a step catalysed by DAHP synthase. Intermediates of the shikimate pathway are 3-dehydroquinate, shikimate, and chorismate (Fig. 1). Phenylalanine is biosynthesised from chorismate, and from phenylalanine all the phenylpropanoids. Quinate is produced from 3-dehydroquinate and incorporated into chlorogenic and isochlorogenic acids (caffeoyl quinic acids) by combination with caffeic acid. Gallic acid is produced from shikimate. 

Plant secondary metabolites are biosynthesized from rather simple building blocks supplied by primary metabolism. Two important metabolic routes in this are the shikimate pathway and the isoprenoid biosynthesis. The shikimate pathway leads to the synthesis of phenolic compounds and the aromatic amino acids phenylalanine, tyrosine and tryptophan. The isoprenoid biosjmthesis is a heavily branched pathway leading to a broad spectrum of compounds (fig. 1). From plants and microorganisms more than 37,000 isoprenoid compounds have been isolated so far [1]. 

Secondary metabolites, produced by pathways derived from primary metabolic routes, are numerous and widespread, especially in higher plants. More than 20,000 were known in 1985 (Hartmann, 1985), and at least 1000 additional compounds, are described each year. In practice, the difference between the primary and secondary metabolites is fuzzy. Plant hormones such as gibberellic acid, indoleace-tic acid (auxin), ethylene, kinetin, and abscisic acid, as well as compounds involved in plant cell wall structure such as cinnamic acid and its polymeric derivative, lignin, are intermediate between primary and secondary metabolism (Birch, 1973). In some instances, compounds normally considered primary metabolites may accumulate in large amounts and behave in a manner usually associated with secondary metabolites. Entities such as shikimic acid and squalene, which initially were considered secondary metabolites, were subsequently shown to be important intermediates in the formation of primary metabolites (phenylalanine, tyrosine and tryptophan, and steroids, respectively). 

Bacteria, fungi, and plants share a common pathway for the biosynthesis of aromatic amino acids with shikimic acid as a common intermediate and therefore named after it—the shikimate pathway. Availability of shikimic acid has proven to provide growth requirements to tryptophan, tyrosine, and phenylalanine triple auxotrophic bacterial strains. Chorismate is also the last common precursor in the aromatic amino acid biosynthetic pathway, but the pathway is not named after it, as it failed to provide growth requirements to the triple auxotrophs. The aromatic biosynthetic pathway starts with two molecules of phosphoenol pyruvate and one molecule of erythrose 4-phosphate and reach the common precursor, chorismate through shikimate. From chorismate, the pathway branches to form phenylalanine and tyrosine in one and tryptophan in another. Tryptophan biosynthesis proceeds from chorismate in five steps in all organisms. Phenylalanine and tyrosine can be produced by either or both of the two biosynthetic routes. So phenylalanine can be synthesized from arogenate or phenylpyruvate whereas tyrosine can be synthesized from arogenate or 4-hydroxy phenylpyruvate. 

Chalcones are the major intermediates of flavonoid biosynthetic pathways they are produced by the condensation of three molecules of malonyl-CoA and a single molecule of 4-coumaryl-CoA. The major precursor malonyl-CoA is derived from citrate, an intermediate product of the TCA cycle. Acetyl-CoA is produced in mitochondria, plastids, peroxisomes, and cytosol via various routes. The cytosolic acetyl-CoA, produced by the multiple subunit enzyme ATP-citrate lyase, is used by acetyl-CoA carboxylase (ACC) to form malonyl-CoA for flavonoid biosynthesis. Another precursor, 4-coumaryl-CoA, is available via the polypropanoid pathway, in which phenylalanine generated via the shikimate and aerogenate pathway is... 

The amino acid phenylalanine is derived from gallic acid, being this compound biosynthesized in the shikimic acid metabolic route. Most of the phenolic compounds from higher plants are also derived from this amino acid, formed in the phenylpropanoid metabolic route, in the cell cytoplasm, being various enzymes involved in this metabolism. Phenylalanine ammonia lyase interacts with phenylalanine forming cinnamic acid, that is, hydrolyzed by citmamate-4-hydroxylase, rendering p-coumaric acid. Different hydroxylations and/or methoxylations, of this... 

The evidence that (- )-shikimic acid plays a central role in aromatic biosynthesis was obtained by Davis with a variety of nutritionally deficient mutants of Escherichia coli. In one group of mutants with a multiple requirement for L-tyrosine, L-phenylalanine, L-tryptophan and p-aminobenzoic acid and a partial requirement for p-hydroxybenzoic acid, (—)-shikimic acid substituted for all the aromatic compounds. The quintuple requirement for aromatic compounds which these mutants displayed arises from the fact that, besides furnishing a metabolic route to the three aromatic a-amino acids, the shikimate pathway also provides in micro-organisms a means of synthesis of other essential metabolites, and in particular, the various isoprenoid quinones involved in electron transport and the folic acid group of co-enzymes. The biosynthesis of both of these groups of compounds is discussed below. In addition the biosynthesis of a range of structurally diverse metabolites, which are derived from intermediates and occasionally end-products of the pathway, is outlined. These metabolites are restricted to certain types of organism and their function, if any, is in the majority of cases obscure. 

Experiments with (2S,3R)- and (2/, 35 )-[3- H]phenylalanines gave tritium retentions of 44% and 24%, respectively (Vederas and Tamm, 1976). Simultaneous incorporation of equal amounts of both enantiomers led to the expected 34% retention of hydrogen label. Transamination occurs stereo-specifically at position 2 of the amino acid therefore, the participation of at least two enzymes with different stereochemical requirements at the 3 position is reasonable. Two biosynthetic pathways are consistent with the data available (see Fig. 13). Path A in Fig. 13 depicts (2 S )-phenylalanine as the actual precursor which is in rapid equilibrium with its enantiomer in path B phenylpyruvic acid (49), derived directly from shikimic acid, is the primary precursor. Considerable suppression of the incorporation of D-amino acid by phenylpyruvic acid (49) indicated that the naturally abundant L-enantiomer is the actual primary precursor, thus demonstrating that path A (Fig. 13) is probably the main biosynthetic route. Both enantiomers are in rapid equilibrium with phenylpyruvic acid (49) via the action of aminotransferases or amino acid oxidases. The stereochemistry of hydrogen loss... 

This route, often called the shikimic acid pathway involves the condensation of phosphoenolpyruvate (2) and a 4-carbon sugar erythrose-4-phosphate (1) which is derived from the pentose phosphate pathway. The product of this reaction is converted to shikimic acid (3). Phosphorylation of shikimic acid to yield 5-phosphoshikimic acid (4) is followed by the addition of another molecule of phospho-enol pyruvate (2) which results in the synthesis of prephenic acid (5). Aromatization of the prephenic acid can give rise to phenylpyruvic acid (6) which upon transamination becomes phenylalanine. The carbon skeletons of the other aromatic amino acids, tryptophane and tyrosine are also synthesised via the shikimic acid pathway as is lignin and many of the aromatic secondary products described in Chapter 6. 

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