Chorismate synthase

3 Chorismate Synthase. - Chorismate synthase catalyses the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh and last enzyme of the shikimate pathway. Chorismate constitutes a major building block for the biosynthesis of an array of aromatic compounds, including the amino acids phenylalanine, tryptophan and tyrosine. Although this reaction does not involve a change in redox states, the enzyme requires reduced FMN for activity, and binds oxidized flavin only very weakly which results in its isolation as the flavin-free apo-enzyme. Macheroux and co-workers have used spectrophotometry, fluorimetry and EPR and ENDOR to investigate binding of the oxidized, reduced and radical forms of FMN to chorismate synthase in the presence of (6R)-6-fluoro-5-enolpyruvylshikimate-3-phosphate(a substrate ana- 

Binding of fully oxidized FMN to enzyme was considerably enhanced in the presence of substrate, while a smaller enhancement was observed with product, leading the authors to suggest that a ternary enzyme-FMN-(substrate/product) complex is formed. Reduction of FMN in the presence of enzyme resulted in the formation of fully reduced, enzyme-bound, flavin. When FMN was reduced in the presence of both enzyme and the substrate analogue (6R)-6-fluoro-5-enol-pyruvylshikimate-3-phosphate a neutral flavin radical was formed. The radical was also formed to a lesser extent in the presence of 5-enolpyruvylshikimate-3-phosphate (40%) and chorismate (14%). 

When 5-enolpyruvylshikimate-3-phosphate was added to an anaerobic sample of reduced FMN and chorismate synthase, the substrate was immediately converted to chorismate, followed by slow formation of a flavin radical. Similarly, when (6R)-6-fluoro-5-enolpyruvylshikimate-3-phosphate was added to an identical sample, rapid formation of the radical was observed. It was suggested that the reduced flavin/enzyme complex is not stable in the presence of substrate or substrate analogue and is oxidized by an unidentified species to yield the flavin radical. 

EPR showed that the radical had g = 2.0039 and a linewidth of 2.1 mT which decreased to 1.5 mT in deuterated buffer, consistent with a neutral flavin radical. The linewidth was independent of whether chorismate or (6R)-6-fluoro-5-enol-pyruvylshikimate-3-phosphate was present. 

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Figure 1. Schematic outline of various products and associated enzymes from the shikimate and phenolic pathways in plants (and some microorganisms). Enzymes (1) 3-deoxy-2-oxo-D-arabino-heptulosate-7-phosphate synthase (2) 5-dehydroquinate synthase (3) shikimate dehydrogenase (4) shikimate kinase (5) 5-enol-pyruvylshikimate-3-phosphate synthase (6) chorismate synthase (7) chorismate mutase (8) prephenate dehydrogenase (9) tyrosine aminotransferase (10) prephenate dehydratase (11) phenylalanine aminotransferase (12) anthranilate synthase (13) tryptophan synthase (14) phenylalanine ammonia-lyase (15) tyrosine ammonia-lyase and (16) polyphenol oxidase. (From ACS Symposium Series No. 181, 1982) (62).

Flavin Mononucleotide (FMN) Methionine synthase reductase, Chorismate synthase... 

BUTYRYLCHOLINE ESTERASE CHOLINE SULFATASE CHOLINE SULFOTRANSFERASE CHOLOYL-OoA SYNTHETASE OHONDROITIN 4-SULFOTRANSFERASE OHONDROSULFATASES CHORISMATE MUTASE CHORISMATE SYNTHASE Chromatin self-assembly,... 

CELLOBIOSE PHOSPHORYLASE CHORISMATE SYNTHASE COBALAMIN ADENOSYLTRANSFERASE 3-DEHYDROQUINATE SYNTHASE... 

Inhibition of Chorismate Synthase Shikimic and quinic acids are used by microorganisms, fungi, and superior plants for the synthesis of essential aromatic amino acids from acyclic sugars. Fluorinated analogues of substrates and reaction intermediates have been synthesized in order to inhibit enzymes involved in... 

Elimination of P from 5-enolpyruvylshikimate 3-P (Eq. 25-3 and Fig. 25-1, step g) produces chorismate.30 The 24-kDa chorismate synthase, which catalyzes this reaction, requires for activity a reduced flavin. Although there is no obvious need for an oxidation reduction coenzyme, there is strong evidence that the flavin may play an essential role in catalysis, perhaps via a radical mechanism.31-331 ... 

Shikimate is further converted to shikimate 3-phosphate (3.22) by shikimate kinase, and subsequently to 5-e o/pyruvylshikimate 3-phosphate (EPSP 3.23) by 5-e o/pyruvylshikimate 3-phosphate synthase. EPSP is then converted to chorismate (3.24) by chorismate synthase. 

Chorismate synthase (CS) catalyzes the formation of chorismate, the last step in the shikimate pathway. Chorismate is a branch-point metabolite used for the synthesis of aromatic amino acids, p-aminobenzoic acid, folate, and other cyclic metabolites such as ubiquinone. The shikimate pathway is found only in plants, fungi, and bacteria, making the enzymes of the pathway potential targets for herbicides, antifungals, and antibiotics. 

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]. 

A considerable amount of effort has gone into the detection of the shikimic acid pathway in plants with the exception of chorismate synthase, all enzymes of the pathway have been demonstrated in plant extracts. Nevertheless there are still gaps in our knowledge of the pathway in plants and apparent anomalies that need to be resolved. These will be detailed in the following discussion. 

With the exception of chorismate synthase [Fig. 2 (7)] all enzymes leading to chorismate synthesis shown in the pathway in Fig. 2 have been demonstrated in plants (Table I). While there is little doubt the pathway shown in Fig. 2 or one very similar to it functions in higher plants there are several pathway relationships yet to be elucidated as will become evident in the discussion which follows. 

No reports have appeared on the detection in plants of chorismate synthase the flnal enzyme in the prechorismate portion of the shikimate pathway. The enzyme has been purified to apparent homogeneity from Bacillus subtilis (Hasan and Nester, 1978). 

Although the formation of p-aminobenzoic acid (36) (Fig. 7.12) can be explained by amination and loss of pyruvate from w<7-chorismic acid, enzyme extracts from Enterobacter aerogenes and two Streptomyces species contain p-amino-benzoate synthase and /5< -chorismate synthase activity. Kinetic data suggest that synthesis of p-aminobenzoic acid occurs from chorismic acid (Johanni et al., 1989). p-Aminobenzoic acid is important in the formation of folic acid in fungi and bacteria (Haslam, 1974). 

PouLSEN, C. and R. Verpoorte, Roles of chorismate mutase iso-chorismate synthase and anthranilate synthase in plants, Phyto-chemistiy, 30, 377-386 (1991). 

Frost and Draths (23) were unsuccessful in their attempt to continue their systematic accumulation of intermediates in the aromatic amino acid biosynthetic pathway when the three enzymes discussed above (DAHP synthase, transketolase, and DHQ synthase) were overexpressed in an E. coli strain lacking chorismate synthase activity. This led to the discovery that under the conditions of their assay, catechol was being produced, along with beta-ketoadipate. The Klebsiella pneumoniae enzymes involved in the conversion of DHS to catechol, DHS dehydratase and protocatechuate decarboxylase, were used since the corresponding E. coli enzymes have not yet been cloned. This discovery may have a significant industrial impact, as these two chemicals have important uses in the chemical industry. Catechol, for example, can be used to produce the flavoring vanillin, as well as L-dopa, epinephrine, and norepinephrine. Adipic acid is used in the production of nylon-6,6. 

Deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase 2 5-dehydroquinate synthase 3 quinate dehydrogenase 4 5-dehydroquinate dehydratase 5 shikimate dehydrogenase 6 shikimate kinase 7 3-enolpyruvylshikimate-5-phosphate synthase 8 chorismate synthase... 

The information obtained from the application of glyphosate to complex systems strongly pointed to one of the following three enzymes as the target of the inhibitor in the shikimate pathway shikimate kinase (EC 2.7.1.71), 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19), and chorismate synthase (EC 4.6.1.4). Jointly, these three enzymes convert shikimic acid to chorismic acid in a series of interesting reactions >(Fig. 2). A defined system" had therefore to be found in which the conversion of shikimic acid to chorismic acid could be conveniently studied. 

Now, in the presence of chorismate synthase (EC 4.2.3.5), 5-(l-carboxyvinyl)-3-phosphoshikimate loses phosphate and the Hr proton at Ce to form the conjugated diene, chorismate. 

Scheme 11.83. The conversion of 5-(l-carboxyvmyl)-3-phosphoshikimate to the conjugated diene, chorismate, in the presence of chorismate synthase (EC 4.2.3.5). It has been argued that this is a free radical-type process using a flavin mononucleotide (FMN) that is reduced in the process (FMNH2). After Osborne, A. Thomeley, R. N. Abell, C Bornemann, S. /. Biol. Chem.,im,275,35825.

Next (cf. Scheme 11.81), the 3-dehydroquinate was shown to undergo dehydration to produce 3-dehydroshikimic acid and reduction (cf. Scheme 11.82) to shiki-mate (shikimate dehydrogenase, EC 1.1.1.25). In the same scheme, a depiction of phosphorylation to 3-phosphoshikimate (shikimate kinase, EC 2.7.1.71) followed and was shown to set the stage for the reaction of the latter with phosphoenol pyruvate under the influence of the enzyme (3-phosphoshikimate 1-carboxyvinyl transferase, EC 2.5.1.19) to yield 5-(l-carboxyvinyl)-3-phosphoshikimate. Finally, with chorismate synthase (EC 4.2.3.5) (Scheme 11.83), the penultimate progenitor of phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W), viz., chorismate, is produced. These processes are shown in detail in Schemes 11.80-11.83 and, in less detail, in Scheme 12.20. 

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