5-Dehydroshikimate accumulation

Quinic acid, a compound accumulated by many green plants, can be formed by reduction of 3-dehy-droquinate (Eq. 25-2) in both plants and bacteria. Quinic acid can be converted into useful industrial products such as benzoquinone and hydroquinone, and its production by bacteria provides a convenient route to these compounds.168 In the main shikimate pathway 3-dehydroquinate is dehydrated to 3-dehydroshikimate (Eq. 25-3). The latter can be dehydrated... 

Intermediates in the biosynthetic route are 3-dehydroshikimic acid, protocate-chuic acid and catechol (Scheme 7.16). Optimization of microbial ds,ds-muconic acid synthesis required the expression of three enzymes not typically found in Escherichia coli [42cj. E. coli WNl/pWN2.248 was developed that synthesized 36.8 g of ds,ds-muconic acid in a 22% (mol/mol) yield from glucose after 48 h of culturing under fed-batch fermentor conditions. Optimization of the carbon flow directed into ds,ds-muconic acid biosynthesis and manipulation of enzyme activities were aimed at avoiding accumulation of biosynthetic intermediates. 

Because of the lack of shikimic acid dehydrogenase, 3-dehydroshikimic acid accumulates. Catechol-0-methyltransferase is not very selective. Apart from methylation in the meta-position, the para-hydroxy group is also methylated to an extent of ca. 20 mole%. The addition of methionine increases significantly the vanillic acid content in the product mixture. 

The products accumulated and isolated from the cultures of mutants are sometimes not those of the actual pathway, but modified products derived from intermediates involved. For example, in Neurospora mutants blocked after the formation of dehydroshikimic acid, only protocatechuic and vanillic acids (derived from dehydroshikimic acid) accumulate. In other instances, an intermediate or a derivative of an intermediate may be converted by a pathway that usually... 

Further studies by Davis on the E. coli mutants that can utilize shikimic acid indicated the presence of three different types type 1 accumulates 5-dehydroshikimic acid type 2 can use this compound and accumulates 5-dehydroquinic acid type 3 can use all three aromatic precursors. There is some evidence that quinic acid may also be involved... 

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... 

After this advance it was observed that mutants blocked before shikimic acid accumulated earlier intermediates. One of these was identified to be 5-dehydroshikimic acid ( 4)- The isolated compound was determined to be a seven-carbon monocarboxylic acid (pif, 3.2) with two acylable hydroxyl groups. The compoimd is levorotatory and has an absorption band in the ultraviolet. Besides the chemical evidence, the fact that the compound could be converted to shikimic acid by mutants that accumulate the latter, strongly supports the conclusion that this intermediate is dehydroshikimic acid. Purification of dehydroshikimic acid could be followed easily siuce it is converted to shikimic acid on autoclaving. 

A third intermediate that accumulated in mutants blocked before dehydroshikimic acid was shown to be 5-dehydroquinic acid ( 5). De-hydroquinic acid was found to be moderately stable in acid and extremely unstable in alkali. On heating in dilute acid (pH 1-5) at 100° it is partly converted to dehydroshikimic acid. The structiu% of dehydroquinic acid was established from its infrared spectra, its chemical characteristics, analytical composition, and its conversion to 5-dehydroshikimic acid. 

An enzyme that converts dehydroquinic acid to dehydroshikimic acid was obtained from wild-type E. eoli or Aerdbactor cells and named 5-de-hydroquinase 21S). This enzyme was absent in auxotrophs that accumulated dehydroquinic acid. No evidence of a cofactor was observed, but the enzyme preparation was too crude for this to be conclusive. 

To test this hypothesis experiments were performed on the conversion of various carbohydrates to 5-dehydroshikimic acid by extracts of E. colt. Cell extracts were chosen to eliminate permeability barriers and dehydro-shikimic acid wtfs selected because the equilibrium between dehydroquinic acid and dehydroshikimic acid favored the accumulation of the latter. 

---