Dehydroquinate pathway isolation

Brief mention has already been made in Section II concerning the conversion of 5-dehydroquinate (X) to 5-dehydroshikimate (IX), and of IX to shikimate. This was the first sequence of the pathway to be elucidated, and it is an excellent illustration of the methods made possible by the use of the penicillin technique for the isolation of bacterial auxotrophs. ... 

Davis concluded that shikimic acid was a common precursor of phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, p-hydroxybenzoic acid, and an unknown sixth factor, and he next set out to determine other substances lying on the biosynthetic pathway. The various mutants were therefore tested for syntrophism, i.e., for the ability of one mutant to produce a substance necessary for the growth of another mutant. There was thus found a thermolabile substance, X, which was a true precursor of shikimic acid (184). X was isolated from culture filtrates and identified as 5-dehydroshikimic acid (744). Similar experiments revealed a substance, W, which was a true precursor of substance X (187, 193). This also was isolated and shown to be 5-dehydroquinic acid (906). The enzyme, named 5-dehydroquinase, converting dehydroquinic acid to dehydroshikimic acid has been partially purified (606). It is fairly stable, has a high specificity, appears to have no cofactors, and is of wide occurrence in bacteria, algae, yeasts, and plants but, as expected, could not be found in mammalian liver. 

The structure of 3-dehydroquinic acid as a cyclic aldol made it appear probable that its six-membered ring is formed from an acyclic precursor through an intramolecular aldoli-zation it would then be the first compound in the pathway in which the ring of phenylalanine, tyrosine, tryptophan, etc., is already present, although not yet aromatic. Not long afterwards, this interpretation was shown to be correct when Sprinson and his coworkers at Columbia University isolated DAHP, the acyclic earliest intermediate, and studied its cyclization to 3-dehydroquinic acid. 

Quinic acid, a carboxylated tetrahydroxycyclohexane, is a secondary plant substance formed from dehydroquinic acid, an intermediate in the shikimic acid pathway, the metabolic pathway leading to aromatic compounds. It, therefore, is ubiquitous in living plant cells. It has been isolated from cinchona bark and detected in the cambial sap of conifers (114). 

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... 

Enzymes involved as catalysts in each of the steps from 3-dehydro-quinate (10) to chorismate (14) in the conunon pathway have all been subsequently isolated and characterised from bacterial mutants. Methods of assay for each form of activity have been described . Mitsuhashi and Davis first isolated 3-dehydroquinate dehydratase (E.C. 4.1.2.10) the enzyme which is responsible for the dehydration step (10 11). With a partially purified extract they showed that the... 

Dehydroquinate synthetase, the enzyme responsible for the cyclisation of DAHP (9) to give 3-dehydroquinate (10), the first cyclic intermediate in the shikimate pathway, was obtmned in partially purified form from Escherichia coli. The enzyme required Co and NAD" " (but not NADP" ) for full activity. No intermediates were isolable when these cofactors were removed but it was observed in a kinetic analysis of the enzymic transformation that the release of orthophosphate, the disappearance of DAHP and the formation of 3-dehydroquinate aU proceeded at the same rate. These observations indicated, it was suggested, that one enzyme was responsible for the whole sequence of reactions necessary for the convosion. On the basis of these observations, Sprinson and his collaborators formulated a working hypothesis for the steps involved in the cyclisation of the substrate DAHP and this is discussed in more detail later. 

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