Aromatic-pathway enzymes

Table III. Response of aromatic-pathway enzymes to mechanical wounding...

Studies of repression control of the synthesis of common aromatic pathway enzymes in other microorganisms such as S. cerevisiae and N. crassa are reviewed by Doy [72], and repression control appears to be lacking or minimal. 

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

A number of conformationally restricted fluorinated inhibitors have been synthesized and evaluated. These smdies show that (1) subtle conformational differences of the substrates affect the inhibition (potency, reversible or irreversible character) (Figure 7.50), (2) a third inhibition process involving an aromatization mechanism could take place (Figure 7.51). When the Michael addition and enamine pathways lead to a covalently modified active site residue, the aromatization pathway produces a modified coenzyme able to produce a tight binding complex with the enzyme, responsible for the inhibition (Figure 7.51). ... 

Synthesis and metabolism of catecholamines. Arrows indicate molecular conversions catalyzed by specific enzymes. Bold arrows indicate major (preferred) pathways. Enzymes (I) tyrosine hydroxylase (2) aromatic L-amino acid decarboxylase (3) dopamine-jSymonooxygenase (4) PNMT (5) cateckel-o-methyltransferase (6) monoamine oxidase. 

Jaworski (4) reported that growth inhibition of both plant and microbes by glyphosate could be reversed by aromatic amino acids. Further work of Amrhein and his coworkers revealed that glyphosate inhibits the shikimate pathway enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (5). This enzyme catalyzes the reaction shown in Figure 1. Glyphosate-treated plant and bacterial cultures accumulate shikimate and/or shikimate 3-phosphate (S3P), confirming that inhibition of EPSPS is at least a part of the in vivo mechanism of action of this herbicide (6, 7). 

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. 

A feedback inhibition has been detected in B. subtilis, using the ferrisiderophore reductase. This enzyme reduces iron from the ferrisiderophore. The rate at which the ferrisiderophore reductase reduces iron from ferrisiderophores may signal the aromatic pathway about the demand for chorismic acid for 2,3-DHBA synthesis [128,129]. The reductase may have a regulatory effect on chorismate synthase activity. Chorismate synthase may have oxidizable sulfhydryl groups that, when oxidized, may slow the synthesis of chorismic acid [128-130]. There seemed to be no repression or inhibitory effect of 2,3-DHBA or SA on its own biosynthesis [78,121]. Also the endproduct mycobactin (sole endproduct) does not inhibit SA biosynthesis [78]. 

Regulation of biosynthesis in the multibranched shikimic acid pathway appears to rely heavily on feedback mechanisms, some of which have been demonstrated both in vivo as well as in vitro. It would seem from the composite of plant studies described herein, which by no means are extensive enough to permit inclusive generalization, that adequate control measures have been observed to account for the maintenance of pool sizes at reasonably low levels by the various metabolites. Furthermore, negative evidence for repression of pathway enzymes tends to emphasize the role of enzyme inhibition and activation in the regulation of aromatic amino acid biosynthesis in higher plants which at the present is depicted in Fig. 6. 

The synthesis of tryptophan in microorganisms is affected at several levels by end-product inhibition. Thus, end-product feedback inhibition partly regulates the synthesis of chorismic acid which is the final product of the common aromatic pathway and serves as a substrate for the first reaction in the tryptophan-synthesizing branch pathway (see Fig. 2). Regulation of the common aromatic pathway was recently reviewed by Doy [72]. The first enzyme of the common aromatic pathway, 3-deoxy-D-flrah/>jo-heptulosonate 7-phosphate synthetase (DAHPS), has been reported to exist as at least three isoenzymes, each specifically susceptible to inhibition by one of the aromatic amino acid end products (tyrosine, phenylalanine, and tryptophan), in E. coli (see reference [3]). It should be noted that many reports have indicated that in E. coli the DAHPS (trp), the isoenzyme whose synthesis is repressed specifically by tryptophan, was not sensitive to end-product inhibition by tryptophan. Recently, however, tryptophan inhibition of DAHPS (trp) activity has been demonstrated in E. coli [3,73,74]. The E. coli pattern, therefore, represents an example of enzyme multiplicity inhibition based on the inhibition specificity of isoenzymes. It is interesting to note the report by Wallace and Pittard [75] that even in the presence of an excess of all three aromatic amino acids enough chorismate is synthesized to provide for the synthesis of the aromatic vitamins whose individual pathways branch from this last common aromatic intermediate. In S. typhimurium, thus far, only two DAHPS isoenzymes, DAHPS (tyr) and DAHPS (phe) have been identified as sensitive to tyrosine and phenylalanine, respectively [76]. 

The common aromatic pathway is subject to repression control of enzyme synthesis as well as the previously described control by end-product inhibition of enzyme activity. Much of the work is reviewed by Gibson and Pittard [3] and Doy [72]. Most of the studies have centered on repression control of DAMPS, the first enzyme in the pathway. The three DAMPS isoenzymes of E. coli are repressed by their specific aromatic amino acids—phenylalanine, tyrosine [107], and tryptophan [108]. In addition there is cross repression of DAMPS (tyr) synthesis by phenylalanine and tryptophan at high concentrations [107,109], and DAMPS (phe) synthesis is cross-repressed by tryptophan [109,110]. 

The enzymes of the common aromatic pathway, other than DAHPS, show relatively little response to repression control in S. typhimurhm [76] and E. coli [108,110] under most test conditions. However, considerable variation in the level of the shikimate kinase of E. coli has been reported [3] for aromatic auxotrophs under certain growth conditions. The possibility that this enzyme may play an important regulatory role is also suggested by the evidence for two chromatographically separable shikimate kinase enzymes in S. typhimurium [119]. The presence of two isoenzymes of shikimate kinase in this organism would explain the apparent lack of occurrence of strains with mutational defects in this enzyme. 

The aromatic biosynthetic pathways do not exist in isolation. Some interactions among the branches of the common aromatic pathway have been mentioned in this chapter. Substrates which are common to several different pathways can serve to interconnect the functioning of the pathways, although they may appear to be quite independent. Jensen has coined the term metabolic interlock to describe regulatory interactions among different metabolic pathways [146a,243,244]. A number of reports have appeared of interactions between the histidine and tryptophan pathways in B. subtilis involving effects on the rates of enzyme synthesis [245,246] as well as enzyme activity [244], Evidence for an interaction between the histidine and tryptophan pathways of N. crassa has also been reported [247],... 

The metabolic pathway responsible for biosynthesis of aromatic amino acids and for vitamin-like derivatives such as folic acid and ubiquinones is a major enzyme network in nature. In higher plants this pathway plays an even larger role since it is the source of precursors for numerous phenylpropanoid compounds, lignins, auxins, tannins, cyano-genic glycosides and an enormous variety of other secondary metabolites. Such secondary metabolites may originate from the amino acid end products or from intermediates in the pathway (Fig. 1). The aromatic pathway interfaces with carbohydrate metabolism at the reaction catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the condensation of erythrose-4-phosphate and PEP to form... 

Table 4. Comparison of key enzymes of the aromatic pathway in extracts prepared from homogenates and from chloroplasts of spinach leaves. 

Stanier and Hayaishi suggest that there are two general pathways for the oxidative catabolism of tryptophan, one through anthranilic acid and catechol, referred to as the aromatic pathway and the other through kynurenic acid, termed the quinoline pathway. These pathways have been established to a reasonable degree of certainty in bacterial enzyme systems, but are not so clearly evidenced in the vertebrate organism. 

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