Shikimate pathway enzyme

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. 

Bode, R. Birnbaum, D. Aggregation and separability of the shikimate pathway enzymes in yeasts. Z. Allg. MikrobioL, 21, 417-422 (1981)... 

Cole al. (72) found that glyphosate increased extracted specific activities of two shikimate pathway enzymes, shikimate dehydrogenase (shikimate NADP oxidoreductase. Figure 3, No. 

There is growing evidence that the shikimate pathway enzymes exist in the chloroplast as well as in the cytoplasm. Shikimate dehydrogenase isoen-... 

In this chapter, the discussion will concentrate on two inhibitors with a reasonable claim to selective action on enz3ones related to the shikimate pathway glyphosate, which inhibits 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase and L-a-aminooxy-3 phenylpropionic acid (L-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL) (Fig. 2). In addition to introducing a novel inhibitor of PAL, (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP), previous and current efforts to design inhibitors of other shikimate pathway enzymes will be described. The treatment presented here will show that the deductions and predictions made on the basis of the abstract scheme in Figure 1 can be, and have been, tested on the basis of the real pathway presented in Figure 2. 

The paucity of information on shikimate pathway enzymes from higher plants, in particular shikimate... 

Fig. 3 Quaternary structures of the (a) fatty acid beta-oxidation complex of E. coli (From [12], (b) porcine fatty acid synthase (From [14]), and (c) active sites of the Arabidopsis shikimate pathway enzymes, DHQ left) and SDH (right) (From [17])...

Ducati RG, Basso LA, Santos DS (2007) Mycobacterial shikimate pathway enzymes as targets for... 

The Shikimate pathway is responsible for biosynthesis of aromatic amino acids in bacteria, fungi and plants [28], and the absence of this pathway in mammals makes it an interesting target for designing novel antibiotics, fungicides and herbicides. After the production of chorismate the pathway branches and, via specific internal pathways, the chorismate intermediate is converted to the three aromatic amino acids, in addition to a number of other aromatic compounds [29], The enzyme chorismate mutase (CM) is a key enzyme responsible for the Claisen rearrangement of chorismate to prephenate (Scheme 1-1), the first step in the branch that ultimately leads to production of tyrosine and phenylalanine. 

Schultz and coworkers (Jackson et a ., 1988) have generated an antibody which exhibits behaviour similar to the enzyme chorismate mutase. The enzyme catalyses the conversion of chorismate [49] to prephenate [50] as part of the shikimate pathway for the biosynthesis of aromatic amino acids in plants and micro-organisms (Haslam, 1974 Dixon and Webb, 1979). It is unusual for an enzyme in that it does not seem to employ acid-base chemistry, nucleophilic or electrophilic catalysis, metal ions, or redox chemistry. Rather, it binds the substrate and forces it into the appropriate conformation for reaction and stabilizes the transition state, without using distinct catalytic groups. 

Aryl side chain containing L-a-amino acids, such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), are derived through the shikimate pathway. The enzymatic transformation of phosphoenolpyr-uvate (PEP) and erythro-4-phosphate, through a series of reactions, yields shikimate (Scheme 2). Although shikimate is an important biosynthetic intermediate for a number of secondary metabolites, this chapter only describes the conversion of shikimate to amino acids containing aryl side chains. In the second part of the biosynthesis, shikimate is converted into chorismate by the addition of PEP to the hydroxyl group at the C5 position. Chorismate is then transformed into prephenate by the enzyme chorismate mutase (Scheme 3). 

To what extent is the response of cytosolic and plastidic isozymes of the shikimate pathway coordinated or coupled with one another and to alterations in expression of enzymes of the flavonoid and phenylpropanoid-pathway segments Some of the emerging information is given in Figure 6. Thus, light induction, well known to induce PAL and enzymes of the flavonoid pathway, also induces both DS-Mn and DS-Co in parsley cell cultures (49). However, only the cytosolic CM-2 (and not the plastidic CM-1) was induced. Fungal elicitor was reported to induce only DS-Mn—not DS-Co or either of the chorismate mutase isozymes (49). Previous studies... 

Applications of the inhibition of enzymes of the shikimic pathway have given rise to the synthesis of numerous fluorinated derivatives of shikimic acid, especially for applications in crop sciences (bactericides, fungicides, herbicides). " Due to the lack of precise data on the inhibition mechanism, these examples are not considered here. 

FIGURE 16.3 Overview of the biosynthesis of (I) chalcones and (II) 6 -deoxychalcones. The sequential condensation of three molecules of malonyl-CoA (acetate pathway) and p-coumaroyl-CoA (shikimate pathway) is catalyzed by the enzyme chalcone synthase.The production of 6 -deoxychalcones is thought to involve an additional reduction step at the tri- or tetraketide level, catalyzed by polyketide reductase.The origin of the A-ring carbons derived from the acetate pathway is indicated in bold. CoA, coenzyme A. 

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). 

There is interest in applying genetic engineering to increase the output of the shikimate pathway for production of industrially important aromatic compounds, e.g., the dye indigo, which is used in manufacture of blue denim (Box 25-C). Study of the enzymes involved has led to the development of potent inhibitors of the shikimate pathway which serve as widely used herbicides.2 3... 

Figure 25-1 Aromatic biosynthesis by the shikimate pathway. The symbols for several of the genes coding for the required enzymes are indicated. Their locations on the E. coli chromosome map are shown in Fig. 26-4. The aminoshikimate pathway which is initiated through 4-aminoDAHP leads to rifamycin and many other nitrogen-containing products.

The enzyme complex that catalyses steps d to/of Fig. 25-20 has an unusual composition. An a3 trimer of 23.5-kDa subunits is contained within an icosahe-dral shell of 60 16-kDa (3 subunits, similar to the protein coats of the icosahedral viruses (Chapter 7). The (3 subunits catalyze the formation of dimethylribityllu-mazine (steps d, e), while the a3 trimer catalyzes the dismutation reaction of step/, the final step in riboflavin formation.365 A separate bifunctional bacterial ATP-dependent synthetase phosphorylates riboflavin and adds the adenylyl group to form FAD.366 Two separate mammalian enzymes are required.367 Synthesis of deazaflavins of methanogens (Fig. 15-22) follows pathways similar to those of riboflavin. However, the phenolic ring of the deazaflavin originates from the shikimate pathway.368... 

The general phenylpropanoid pathway begins with the deamination of L-phenylalanine to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL), Fig. (1), the branch-point enzyme between primary (shikimate pathway) and secondary (phenylpropanoid) metabolism [5-7]. Due to the position of PAL at the entry point of phenylpropanoid metabolism, this enzyme has the potential to play a regulatory role in phenolic-compound production. The importance of this is illustrated by the high degree of regulation both during development as well as in response to environmental stimuli. 

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