Escherichia coli shikimate pathway

This pattern of labelling, when it was analysed, prompted Sprinson to propose that in Escherichia coli (—)-shikimic add was biosynthesised from a three carbon fragment of glycolysis (8) and a four carbon sugar (7) which was derived from the pentose phosphate pathway of carbohydrate metabolism. 

FIGURE 3.2 The common aromatic pathway to chorismate in Escherichia coli K12, where 5 is phosphoe-nolpyruvate, 6 is erythrose 4-phosphate, 7 is 3-deoxy-D-arabinoheptulose 7-phosphate, 8 is 3-dehydroquinic acid, 9 is 3-dehydroshikimic acid, 10 is shikimic acid, 11 is shikimic acid 3-phosphate, and 12 is 5-enolpyru-vylshikimic acid 3-phosphate. 

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... 

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. 

The first cyclic intermediate on the shikimate pathway is 3-dehydro-quinic acid (38) and it was first obtained by Weiss, Davis and Mingioli from cultures of Escherichia coli mutant 170-27 by charcoal chromatography and precipitation of the brucine salt. Probably the first chemical preparation of this biochemical intermediate was reported by Hesse in 1859 who oxidised (—)-quinic acid (42) with bromine water Several procedures have since been reported for the preparation of this metabolite Yields of almost 80 per cent of 3-hydroquinic acid (38) were reported by Whiting and Coggins after a seven-day incubation of (—)-quinic acid (42) with Acetomonas oxydans CR-49 and similar selective oxidation of the axial 3-hydroxyl group in (—)-quinic acid with nitric acid or platinum and oxygen " forms the basis of chemical methods of preparation. 

The evidence that (- )-shikimic acid plays a central role in aromatic biosynthesis was obtained by Davis with a variety of nutritionally deficient mutants of Escherichia coli. In one group of mutants with a multiple requirement for L-tyrosine, L-phenylalanine, L-tryptophan and p-aminobenzoic acid and a partial requirement for p-hydroxybenzoic acid, (—)-shikimic acid substituted for all the aromatic compounds. The quintuple requirement for aromatic compounds which these mutants displayed arises from the fact that, besides furnishing a metabolic route to the three aromatic a-amino acids, the shikimate pathway also provides in micro-organisms a means of synthesis of other essential metabolites, and in particular, the various isoprenoid quinones involved in electron transport and the folic acid group of co-enzymes. The biosynthesis of both of these groups of compounds is discussed below. In addition the biosynthesis of a range of structurally diverse metabolites, which are derived from intermediates and occasionally end-products of the pathway, is outlined. These metabolites are restricted to certain types of organism and their function, if any, is in the majority of cases obscure. 

In 1950, Davis showed that p-hydroxybenzoic acid had vitamin-Uke activity for multiple aromatic auxotrophs of Escherichia coli but it was not until 19W that Gibson and Cox showed that this growth factor requirement was associated with the formation of ubiquinones in these mutant organisms. Subsequent work has revealed that p-hydroxybenzoic acid (12), a derivative of the shikimate pathway, is a focal metabolite for ubiquinone biosynthesis in a very wide range of organisms - Thus [ C]-p-hydroxybenz-... 

The usual method of study is to suggest a possible precursor and to feed it to the biosynthesizing system. The precursor has to be labelled in some way to trace it through the sequence of reactions, and that is usually by some isotopic element. It may be a radio-active isotope, such as H, " 0, or that can be followed by its radiation or it can be a stable heavy isotope, such as H, C, N, or 0, that can be traced by mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy (Table 5.1). Another possible way is to use mutant strains of an organism that lack the enzymes to complete a particular synthesis, or to add a specific enzyme inhibitor, so that intermediates accumulate and can be identified. A mutant strain of yeast was important in discovering mevalonic acid and its place in terpene biosynthesis (Chapter 6) and a number of mutants of the bacterium Escherichia coli helped to understand the shikimic acid pathway (Chapter 8). 

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