Chorismate mutants

A Try mutant would not be subject to feedback inhibition by overproduction of tryptophan. Also, the mutation may allow more chorismate to proceed to prephenate via E3 (see Figure 8.4) and thus through to L-phenylalanine. 

Chorismate mutase (CM) catalyzes conversion of chorismate into prephenate in a Claisen rearrangement prephenate is a precursor in the biosynthesis of both i-phenylalanine (i-Phe) and i-tyrosine (i-Ttyr). CM occurs as a dimer for structural studies, the monomer was needed (MacBeath, 1998). The assay for CM activity was based on growth of the colonies in the absence of i-Phe and L-Tyr and thus was based on selection, not screening. One resulting mutant was found to be a monomer and to contain a somewhat polar Ala-Arg-Trp-Pro-Trp-Ala sequence. 

Hur S, TC Bruice (2003c) Just a near attack conformer for catalysis (chorismate to prephenate rearrangements in water, antibody, enzymes, and their mutants). J. Am. Chem. Soc. 125 (35) 10540-10542... 

The exbB mutants (47) are derepressed in enterobactin synthesis and produce this siderophore in iron-containing media. The means whereby iron represses enterobactin synthesis is still obscure. Several years ago it was noted that growth of E. coli on low iron media led to changes in various fRNAs (77). In E. coli K-12 aromatic amino-acid synthesizing enzymes are also derepressed in low iron media, possibly because of the diversion of the chorismate pool to enterobactin (78). 

For the aromatic pathway (Figure 30.20), the critical control points are the condensation of phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate 7-phosphate, DAHP, by DAHP synthase. For tryptophan, the formation of anthranilic acid from chorismic acid by anthranilate synthase is the second critical control point. The transcriptional regulation was overcome through the use of alternative promoters and allosteric regulation was circumvented by the classical technique of selection for feedback-resistant mutants using toxic analogues of the repressing compounds. 

MK biosynthesis by the OSB pathway has been elucidated on the basis of isotopic tracer experiments, isolation of mutants blocked in the various steps, isolation and identification of intermediates accumulated by the mutants, and by enzyme assays. Early isotopic tracer experiments with various bacteria established that methionine and prenyl PPi contribute to the methyl and prenyl substituents of the naphthoquinone. The early isotopic tracer studies and other work have been reviewed by Bentley and Meganathan. " In 1964, Cox and Gibson observed that [G- " C] shikimate was incorporated into both MK and ubiquinone by E. coli, thus providing the first evidence for the involvement of the shikimate pathway." Chemical degradation of the labeled isolated menaquinone (MK-8) showed that essentially all of the radioactivity was retained in the phthalic anhydride. It was concluded that the benzene ring of the naphthoquinone (sic) portion of vitamin K2 arises from shikimate in E. coli The authors further suggested that shikimate was first converted to chorismate before incorporation into MK. A more complete chemical degradation of the MK derived from... 

In a subsequent study, by chemical mutagenesis, a number of MK auxotrophs were isolated. These auxotrophs required the incorporation of either MK-4 or chorismate into the medium for growth. Furthermore, these mutants failed to respond to shikimate, thus establishing that the branch point of the pathway is at chorismate." ... 

Prephenlc Acid, l-Carboxy.4-hydroxy-< -oxo-2.5-cyclohexadiene-1-propanoic acid l-carboxy-4-hydraxy-2,S-cyclohexadiene-l-pyruvic acid, C,0H1 Ot mol wt 226.18. C 53.10%, H 4.46%, O 42.44%. Non-aromatic biosynthetic in -termediate that represents a secondary branch-point in the pathway from chorismic acid to phenylalanine and tyrosine, q.q.y., in many organisms. Isoln from cultures of mutant Escherichia coti B. D. Davis, Science 118, 251 (1953). 

Standard microbial methods for investigating tryptophan biosynthesis were applied to plants in order to study auxin biosynthesis [6,212,218]. A series of Arabidopsis mutants with lesions in four sites in the pathway from chorismate to tryptophan has been obtained, but so far no comparable array of mutations exists for any other plant species [18]. Normanly et al. [44] used these Arabidopsis mutants to dissect lAA biosynthesis and showed that the non-tryptophan pathway to lAA branches from tryptophan biosynthesis at the point of indole or indole-3-glycerol phosphate. 

When a P. aeruginosa mutant (PALS 128) was grown under iron rich conditions, the specific activity of the SA-forming enzymes was below the limits of detection [79]. Liu et al. [88], suggest that entC gene expression may be limited at the translational level as well, even when the operon is induced under iron deficiency. This may be understandable because chorismic acid is an essential metabolite for Phe, Trp, Tyr, folate and ubiquinone synthesis. In B. subtilis it was shown that the accumulation of 2,3-DHBA(Glycine) was influenced by the levels of aromatic amino acids and anthranilic acid. Anthranilic acid inhibited the synthesis of DHBA from chorismic acid [117]. It seemed that the reduction in phenolic acid accumulation caused by aromatic amino acids is a consequence of enzyme repression [121]. The synthesis of 2,3-DHBA in B. subtilis is also reduced by other phenolic acids, such as m-substimted benzoic acids. Inhibition of accumulation of phenolic acid by other phenolic acids, would indicate a fairly specific effect on phenolic acid synthesis, but not on the accumulation of coproporphyrin that also accumulates in iron-deficient cultures oiB. subtilis [121]. 

In B. subtilis, the pathway from chorismate to tryptophan is feedback-inhibited by tryptophan, which suppresses anthranilate synthase activity. Mutant B. subtilis that lacks tryptophan synthetase can grow on minimal medium only when supplemented with exogenous tryptophan. Under these conditions, none of the intermediates in the tryptophan biosynthetic pathway from anthranilate to indole 3-glycerol phosphate are produced. However, when the bacteria have depleted the medium of tryptophan, the levels of those intermediates increase, even though there is no net production of tryptophan. Why ... 

Use of Mutants in Biosynthetic Studies Formation of Chorismic Acid Derivatives of Chorismic Acid Biosynthesis of Tryptophan Indole 3-Acetic Acid Avenalumins from Oats DIMBOA and Related Compounds Biosynthesis of Phenylalanine and Tyrosine Compounds Derived from Shikimic Pathway Intermediates... 

In the aromatic amino acid biosynthesis, chorismic acid (51) is converted into anthranilic acid, which would be a potential precursor providing both nitrogens for the ring as well as the aromatic system of phenazines. However, anthranilic acid and other proposed intermediates like quinic acid, tryptophan, tyrosine, and phenylalanine have been questioned on the basis of studies of mutants of phenazine producing organisms with blocked catabolism of these various possible intermediates. 

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