Chorismate isolation

Using the transition-state analog shown on p. 485 a catalytic antibody with chorismate mutase activity was isolated. Many antibodies catalyzing additional reactions have also been found. Although they are usually less active than natural enzymes, in some cases they approach enzymatic rates. Furthermore, they may catalyze reactions for which no known enzymes exist.h... 

Compound 2 was efficiently isolated by anion-exchange chromatography on Dowex 1x8 resin in a purification process similar to that devised for small amounts of chorismate [14]. Elution of the product occurs under moderately acidic conditions, enabling isolation without aromatization. This procedure enables an isolation of 2,3-trans-CHD on the high decagram scale with a purity of 95% after lyophilization [12]. 

Thirty-four naturally occurring compounds that incorporate the oxazole moiety have been isolated thus far. The sources are diverse—plants of the families Gramineae and Rutaceae, nudibranch egg masses, and microorganisms, the latter having furnished the majority of the compounds. With three exceptions, the marine and bacterial oxazoles appear to have been formed from peptides of aliphatic amino acids, while the oxazoles of the Gramineae and Rutaceae arise from the chorismic acid-phenylalanine pathway. The oxazoles have not been... 

True Darwinian evolution involves multiple cycles of mutation and selection. This process can be mimicked in a laboratory setting to optimize the properties of an inefficient enzyme. The hexameric but weakly active chorismate mutase [95] described in Section 3.3.4.1 has been improved in this way [99]. Mutations were introduced into the gene encoding the hexamer subunit by DNA shuffling (Fig. 3.16) [5, 100], which mimics sexual recombination in vitro. Improved variants were selected, as before, by their ability to complement the chorismate mutase deficiency in bacteria. Plasmid DNA was isolated from the fastest growing cells and the entire procedure was repeated. 

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." ... 

Isochorismate synthase, that might be involved in the biosynthesis of 2,3-DHBA, has recently been purified from Catharanthus roseus cell-suspension cultures and subsequently its gene was cloned (L. van Tegelen, P. Moreno, A. Croes, G. Wullems and R. Verpoorte, submitted for publication). Two isoforms of the enzyme were purified and characterized. Both have an apparent molecular mass of 65 kD. The Km values for chorismic acid are 558 pM and 319 p.M for isoform I and II respectively. The enzymes are not inhibited by aromatic amino acids and require Mg for enzyme activity. The isolated cDNA encodes a protein of 64 kD with a A-terminal chloroplast targeting signal. The deduced amino acid sequence shares homology with bacterial isochorismate synthases, and also with anthranilate synthases, another chorismate utilizing enzyme. 

Studies with isolated enzymes in vitro reveal feedback inhibition of chorismate mutase by phenylalanine and tyrosine. Tryptophan apparently controls its own synthesis by feedback inhibition of anthranilate synthase and furthermore exerts control in the partitioning of chorismate between the two competing routes of chorismate metabolism by its ability to both activate chorismate mutase and relieve the inhibition imposed on this step by phenylalanine and tyrosine. In addition, carbon flux throuch chorismate to prephenate is also sensitive to fluctuations in chorismate concentration due to the allosteric substrate activation of chorismate mutase by chorismate. 

A shikimate-chorismate biogenesis can be more confidently predicted for 6-nitro-wo-vanillic acid (243), a constituent of an as yet unidentified Australian toadstool belonging to Cortinarius 280), The compound (243), the only example of a nitroaromatic metabolite from Cortinarius, produces an intensely yellow dianion [UV/vis. (ethanol-h alkali) max(l g ) = 270.5 (4.53), 429 nm (4.76)] which is responsible for the bright yellow colour of the cut flesh of this fungus. A yellow nitro compound, 3,5,6-trichloro-l, 4-dimethoxy-2-nitrobenzene, has been isolated from Phellinus rimosus = Pomes robiniae) 147). 

Phenazines compounds based on the phenazine ring system (Table). All known naturally occurring P. are produced only by bacteria, which excrete them into the growth medium. Both six-membered carbon rings of P. are biosynthesized in the shikimate pathway of aromatic biosynthesis, via chorismic acid (not from anthranilate, as reported earlier). The earliest identified biosynthetic intermediate after chorismate is phenazine 1,6-dicarboxylate, which has been isolated from Pseudomonas phenazinium and from non-... 

Research in David Sprinson s laboratory at Columbia University, in part contemporary with the research outlined in the preceding pages, has unraveled the earliest stages of the sequence and the derivation of the first, non-cyclic intermediate, DAMP. In the same laboratory, Judith Levin discovered 5-enolpyruvylshikimate 3-phosphate and predicted the existence and correct structure of the next, and last one, of the common intermediates, the compound from which the pathways to the individual primary aromatic products branch off. Isolation and structure proof of this branchpoint intermediate, chorismic acid, by the Australian workers F. Gibson, L.M. Jackman, and J.M. Edwards completed the elucidation of the general pathway. 

In one experiment washed chloroplasts were isolated and assayed for nitrite reductase, DAHP synthase-Mn and chorismate mutase-1 activities (Table 3). Since enzymes may fractionate with organelles by non-specific (or specific) association with the organelle surface, latency determinations were made. With this approach, activity determinations are made before and after rupture of the washed chloroplasts. If activities are located within the organelle, they are expected to increase dramatically following organelle disruption. Thus, nitrite reductase (chloroplast marker enzyme) gave a latency value of 16, a value similar to those obtained for DAHP synthase-Mn and chorismate mutase-1. The identity of chorismate mutase as the CM-1 isozyme was confirmed by its sensitivity to inhibition by L-tyrosine. 

Fig. 8. Ability of analogs of L-tyrosine (left chart) and of L-phenylalanine (right chart) to inhibit chorismate mutase-1 isolated from N. silvestris. ...

Hydroxybenzoic acid 5.35) has been shown to stand as the key intermediate in ubiquinone biosynthesis, in living systems from micro-organisms to mammals. In animals, phenylalanine and tyrosine serve as precursors, but in bacteria chorismic acid 5.10) is the precursor [28, 29]. Interlocking evidence obtained from bacteria in experiments with mutants (and genetic analysis), cell-free preparations, and isolation and identification of intermediates allows clear delineation [25, 29] of the sequence of biosynthesis as that shown in part in Scheme 5.5 beyond 5.35) the intermediates are the... 

Gibson, M. I. and Gibson, F. (1964) Preliminary studies on the isolation and metabolism of an intermediate in aromatic biosynthesis chorismic acid. Biochem. J. 5H), 248-252. 

Mutant strains of Escherichia coli and Aerobacter aerogenes were described which had a quintuple requirement of aromatic substrates (L-phenylalanine, L-tyrosine, L-tryptophan, 4-amino-benzoate and 4-hydroxybenzoate) for growth. Certain of these mutants were found to accumulate (—)-shikimic acid (4) in their culture filtrates and other mutants, blocked in earlier reactions in the pathway, were able to utilise (—)-shikimic acid (4) to replace the aromatic sutetrates. These observations established with great probability that (—)-shikimic add was a common precursor for each of these aromatic compounds. Experiments of this type permitted each of the intermediate in the common pathway, 3-dehydroquinic add (10), 3-dehydroshikimic add (11), (—)-shikimic add (4), shikimic add-3-phosphate (12), 5-enolpyruvylshikimic add-3-phosphate (13) and chorismic acid (14), to be isolated and characterised and for the pathway... 

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