3-dehydroshikimate Research Articles

Structural and Biochemical Analyses of Shikimate Dehydrogenase AroE from Aquifex aeolicus: Implications for the Catalytic Mechanism,

The shikimate biosynthetic pathway is essential to microorganisms, plants, and parasites but absent from mammals. Therefore, shikimate dehydrogenase (SD) and other enzymes in the pathway are attractive targets for developing nontoxic antimicrobial agents, herbicides, and antiparasite drugs. SD catalyzes the fourth reaction in the pathway, the nicotinamide adenine dinucleotide phosphate- (NADP-) dependent reduction of 3-dehydroshikimic acid to shikimic acid (SA), as well as its reverse, by the transfer of a hydride. Previous structural studies reveal that the enzyme exists in two major conformations, an open and a closed form. For the reaction to occur, it is believed that the catalytic complex assumes the closed conformation. Nonetheless, the only structure containing both SA and NADP+ exhibits an open conformation (PDB entry 2EV9). Here, we present two crystal structures of Aquifex aeolicus SD, including a ternary complex with both SA and NADP+, which assumes the closed conformation and therefore contains a catalytically competent active site. On the basis of preexisting and novel structural and biochemical data, a catalytic mechanism is proposed.

Directed Evolution of 2-Keto-3-deoxy-6-phosphogalactonate Aldolase To Replace 3-Deoxy-d-arabino-heptulosonic Acid 7-Phosphate Synthase

Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbial synthesis of shikimate pathway products provides an alternate strategy to circumvent the competition for phosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and the phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGal aldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, and multiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-fold improvement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the addition of pyruvate to d-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2 contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression of NR8.276-2 in the DAHP synthase and shikimate dehydrogenase-deficient E. coli strain NR7 under fed-batch fermentor-controlled cultivation conditions resulted in synthesis of 13 g/L 3-dehydroshikimic acid in 6.5% molar yield from glucose. Increased coexpression of the irreversible downstream enzyme 3-dehydroquinate synthase increased production of 3-dehydroshikimic acid to 19 g/L in 9.7% molar yield from glucose. Coamplification with transketolase, which increases d-erythrose 4-phosphate availability, afforded 16 g/L 3-dehydroshikimic acid in 8.5% molar yield.

Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate‐limited and carbon‐limited conditions

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon- and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult.

Antioxidant activity of 3-dehydroshikimic acid in liposomes, emulsions, and bulk oil.

The antioxidant activity of 3-dehydroshikimic acid (DHS), an intermediate in the biosynthesis of aromatic amino acids, was evaluated in three assay systems: bulk oil (lard), liposomes, and a 10% corn oil-in-water emulsion. Upon initiation of peroxidation in the liposome or emulsion systems, DHS exhibited weak antioxidant activity. In contrast, DHS displayed strong antioxidant activity in lard, suppressing peroxidation with activity comparable to that of tert-butylhydroquinone, propyl gallate, and gallic acid and superior to that of alpha-tocopherol. Two major DHS oxidation products, gallic acid and protocatechuic acid, were identified by gas chromatography/mass spectral analysis of lard extracts; both compounds are effective antioxidants in the bulk oil system. In the liposome system, DHS remained intact throughout the assay period. A small amount of gallic acid was observed in extracts of the emulsion; however, protocatechuic acid was not detected. A mechanism to explain the different activities of DHS in the three lipid systems is proposed.

Gallic acid and hydrolysable tannins are formed in birch leaves from an intermediate compound of the shikimate pathway

Gallic acid is the starting material for hydrolysable tannin synthesis, but the mechanism of its formation in higher plants has not been known. To elucidate the pathway of gallic acid synthesis in the leaves of mountain birch ( Betula pubescens ssp. czerepanovii), we studied the effects of glyphosate (N-[phosphonomethyl]-glycine) on the levels of individual hydrolysable tannins. Glyphosate is known to block 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme of the shikimate pathway, thus reducing the synthesis of aromatic amino acids and phenolics derived from phenylalanine. However, when birch leaves were treated with glyphosate (1 mM, 72 h), the contents of individual hydrolysable tannins increased about twofold compared to control leaves. Our findings indicate that the immediate precursor of gallic acid may be an intermediate compound of the shikimate pathway before EPSP, most probably 3-dehydroshikimic acid. Consistently with this assumption, a highly active enzyme dehydroshikimate dehydrogenase (DSDG), which catalyses the direct conversion of 3-dehydroshikimic acid into gallic acid, was found in birch leaves. This new enzyme was partially purified to study its properties. It was found that DSDG is an NADP-dependent enzyme with a pH optimum at about 10.0. The enzyme showed an affinity for NADP + approximately 60-fold (K m=0.008 mM) that for dehydroshikimate (K m=0.49 mM). The intracellular organisation of the shikimate pathway and the hydrolysable tannin pathway are discussed.

Modulation of phosphoenolpyruvate synthase expression increases shikimate pathway product yields in E. coli.

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed-batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3-dehydroshikimic acid. Shikimate pathway byproducts 3-deoxy-D-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3-dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3-dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.

Hydroaromatic equilibration during biosynthesis of shikimic acid.

The expense and limited availability of shikimic acid isolated from plants has impeded utilization of this hydroaromatic as a synthetic starting material. Although recombinant Escherichia coli catalysts have been constructed that synthesize shikimic acid from glucose, the yield, titer, and purity of shikimic acid are reduced by the sizable concentrations of quinic acid and 3-dehydroshikimic acid that are formed as byproducts. The 28.0 g/L of shikimic acid synthesized in 14% yield by E. coli SP1.1/pKD12.138 in 48 h as a 1.6:1.0:0.65 (mol/mol/mol) shikimate/quinate/dehydroshikimate mixture is typical of synthesized product mixtures. Quinic acid formation results from the reduction of 3-dehydroquinic acid catalyzed by aroE-encoded shikimate dehydrogenase. Is quinic acid derived from reduction of 3-dehydroquinic acid prior to synthesis of shikimic acid? Alternatively, does quinic acid result from a microbe-catalyzed equilibration involving transport of initially synthesized shikimic acid back into the cytoplasm and operation of the common pathway of aromatic amino acid biosynthesis in the reverse of its normal biosynthetic direction? E. coli SP1.1/pSC5.214A, a construct incapable of de novo synthesis of shikimic acid, catalyzed the conversion of shikimic acid added to its culture medium into a 1.1:1.0:0.70 molar ratio of shikimate/quinate/dehydroshikimate within 36 h. Further mechanistic insights were afforded by elaborating the relationship between transport of shikimic acid and formation of quinic acid. These experiments indicate that formation of quinic acid during biosynthesis of shikimic acid results from a microbe-catalyzed equilibration of initially synthesized shikimic acid. By apparently repressing shikimate transport, the aforementioned E. coli SP1.1/pKD12.138 synthesized 52 g/L of shikimic acid in 18% yield from glucose as a 14:1.0:3.0 shikimate/quinate/dehydroshikimate mixture.

Synthesis of gallic acid: Cu(2+)-mediated oxidation of 3-dehydroshikimic acid.

With the elaboration of high-yielding, high-titer syntheses of 3-dehydroshikimic acid from glucose using recombinant Escherichia coli, oxidation of this hydroaromatic becomes a potential route for synthesis of gallic acid. Conversion of 3-dehydroshikimic acid into gallic acid likely proceeds via initial enolization of an alpha-hydroxycarbonyl and oxidation of the resulting enediol. 3-Dehydroshikimate enolization in water was catalyzed by inorganic phosphate while Zn(2+) was used to catalyze enolization in acetic acid. Enediol oxidation employed Cu(2+) as either the stoichiometric oxidant or as a catalyst in the presence of a cooxidant. Gallic acid was produced in a yield of 36% when 3-dehydroshikimic acid in phosphate-buffered water reacted for 35 h with H2O2 and catalytic amounts of CuSO(4). 3-Dehydroshikimate-containing, phosphate-buffered culture supernatants reacted with stoichiometric amounts of CuCO(3)Cu(OH)(2) and Cu(x)(H(3-x)(PO4)(2) to give gallic acid in yields of 51% in 5 h and 43% in 12 h, respectively. Solutions of 3-dehydroshikimic acid in acetic acid reacted with stoichiometric amounts of Cu(OAc)(2) to afford a 74% yield of gallic acid in 36 h. Acetic acid solutions of 3-dehydroshikimic acid could also be oxidized by air using catalytic quantities of Cu(OAc)(2). ZnO accelerated these oxidations leading to a 67% yield of gallic acid in 4 h when an acetic acid solution of 3-dehydroshikimic acid was reacted with O(2) and a catalytic amount of Cu(OAc)(2).

A synthesis of l-α-phosphatidyl- d- myo-inositol 4,5-bisphosphate (4,5-PIP 2) and glyceryl lipid analogs

The title bioactive phosphatidylinositide and short-chain glyceryl lipid analogs were prepared from deoxyinosose 2, which was ultimately derived from 3-dehydroshikimic acid.

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinantEscherichia coli

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.

Fed‐batch fermentor synthesis of 3‐dehydroshikimic acid using recombinant Escherichia coli

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 61–73, 1999.

Cloning and analysis of the shiA gene, which encodes the shikimate transport system of Escherichia coli K-12

In Escherichia coli K-12, the shiA gene is involved in the uptake of shikimate. This gene has been cloned and its nucleotide sequence determined. The gene is predicted to encode a protein of 438 amino acids and lies adjacent to the amn gene. The hydropathy profile and the amino acid sequence indicate that the ShiA protein is a polytopic membrane protein that shows a homology with members of the major facilitator superfamily of transport proteins. Recombining an inactive form of the cloned gene into the chromosome creates mutants unable to transport shikimate. Introducing a wild-type gene on a multicopy plasmid into a shiA mutant restores the ability to transport shikimate. When this multicopy shiA plasmid is introduced into an aroE strain, this strain is now able to grow with shikimate as the aromatic supplement, consistent with the notion that dehydroshikimate (DHS) accumulated in an aroE strain prevents uptake of shikimate by competition. Expression of the shiA gene does not appear to be regulated by the TyrR protein, a repressor/activator that controls the expression of other genes involved with the biosynthesis or transport of the aromatic amino acids.

Control of metabolic flux through the quinate pathway in Aspergillus nidulans.

The quinic acid ulitization (qut) pathway in Aspergillus nidulans is a dispensable carbon utilization pathway that catabolizes quinate to protocatechuate via dehydroquinate and dehydroshikimate(DHS). At the usual in vitro growth pH of 6.5, quinate enters the mycelium by means of a specific permease and is converted into PCA by the sequential action of the enzymes quinate dehydrogenase, 3-dehydroquinase and DHS dehydratase. The extent of control on metabolic flux exerted by the permease and the three pathway enzymes was investigated by applying the techniques of Metabolic Control Analysis. The flux control coefficients for each of the three quinate pathway enzymes were determined empirically, and the flux control coefficient of the quinate permease was inferred by use of the summation theorem. There measurements implied that, under the standard growth conditions used, the values for the flux control coefficients of the components of the quinate pathway were: quinate permease, 0.43; quinate dehydrogenase, 0.36; dehydroquinase, 0.18; DHS dehydratase, <0,03. Attempts to partially decouple quinate permease from the control over flux by measuring flux at pH 3.5 (when a significant percentage of the soluble quinate is protonated and able to enter the mycelium without the aid of a permease) led to an increase of approx. 50% in the flux control coefficient for dehydroquinase. Taken together with the fact that A. nidulans has a very efficient pH homeostasis mechanism, these experiments are consistent with the view that quinate permease exerts a high degree of control over pathway flux under the standard laboratory growth conditions at pH 6.5. The enzymes quinate dehydrogenase and 3-dehydroquinase have previously been overproduced in Escherichia coli, and protocols for their purification published. The remaining qut pathway enzyme DHS dehydratase was overproduced in E. coli and a purification protocol established. The purified DHS dehydratase was shown to have a K(m) of 530 microM for its substrate DHS and a requirement for bivalent metal cations that could be fulfilled by Mg(2+), Mn(2+) or Zn(2+). All three qut pathway enzymes were purified in bulk and their elasticity coefficients with respect to the three quinate pathway intermediates were derived over a range of concentrations in a core tricine/NaOH buffer, augmented with necessary cofactors and bivalent cations as appropriate. Using these empirically determined relative values, in conjunction with the connectivity theorem, the relative ratios of the flux control coefficients for the various quinate pathway enzymes, and how this control shifts between them, was determined over a range of possible metabolic concentrations. These calculations, although clearly subject to caveates about the relationswhip between kinetic measurements in vitro and the situation in vivo, were able to successfully predict the hiearchy of control observed under the standard laboratory growth conditions. The calculations imply that the hierarchy of control exerted by the quinate pathway enzymes is stable and relatively insensitive to changing metabolite concentrations in the ranges most likely to correspond to those found in vivo. The effects of substituting the type I 3-dehydroquinases from Salmonella typhi and the A. nidulans AROM protein (a pentadomain protein catalysing the conversion of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate into 5-enolpyruvylshikimate 3 phosphate), and the Mycobacterium tuberculosis type II 3-dehydroquinase, in the quinate pathway were investigated and found to have an effect. In the case of S. typhi and A. nidulans, overproduction of heterologous dehydroquinase led to a diminuation of pathway flux caused by a lowering of in vivo quinate dehydrogenase levels increased above those of the wild type. We speculate that these changes in qu

Reaction of 3-Dehydroshikimic Acid with Molecular Oxygen and Hydrogen Peroxide: Products, Mechanism, and Associated Antioxidant Activity

In the presence of molecular oxygen or hydrogen peroxide, inorganic phosphate catalyzes the conversion of 3-dehydroshikimic acid (DHS) into gallic acid. Other products formed in the reaction of DHS with oxygen include protocatechuic acid, tricarballylic acid, and pyrogallol. With hydrogen peroxide as oxidant, pyrogallol formation is not observed, and smaller amounts of tricarballylic acid are produced. Evidence favoring a mechanism involving phosphate-catalyzed tautomerization of DHS to a reactive enediol intermediate follows from the successful isolation of dihydrogallic acid when oxidant is excluded from phosphate-buffered solutions of DHS. The reductone-like solution chemistry of DHS and the radical quenching reactivity of the phenolics formed from DHS prompted an appraisal of DHS antioxidant activity. Based on two different analyses, DHS was discovered to possess significant antioxidant activity relative to α-tocopherol, gallic acid, propyl gallate, and tert-butylhydroquinone.

Specificity of E. Coli shikimate dehydrogenase towards analogues of 3-dehydroshikimic acid

Analogues of 3-dehydroshikimic acid which lack the C-4 and C-5 hydroxyl groups have been synthesised and assayed as substrates for shikimate dehydrogenase. The presence of the C-4 hydroxyl group is found to be very important for specificity, whereas the C-5 hydroxyl group is not. The enzyme exhibits enantioselectivity at C-1 and C-4 of the racemic substrate analogues.

A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. II. Isolation and characterization of a shikimate excreting auxotrophic mutant of Nocardia mediterranei with normal rifamycin-production.

The mutant under study, designated A10, is derived from a Nocardia mediterranei strain, N813, which is a high rifamycin B producer. A10 is auxotrophic for aromatic amino acids but unlike A8 (see preceding paper) produces the same amount of rifamycin B as the parent. Shikimic acid and 3-dehydroshikimic acid are accumulated in the fermentation broth of this mutant. It was shown to be blocked in one of the enzymes leading from shikimate to chorismate. No formation of shikimate-3-phosphate from shikimate and ATP could be detected in vitro using crude extracts of this mutant and of the parent. As mutant A10 is only defective in the biosynthesis of aromatic amino acids and not in the biosynthesis of rifamycins it would appear that the seven-carbon amino unit of the rifamycin-chromophore must be derived from an intermediate of the shikimate pathway not behind shikimate. By referring to the results of the preceding paper it can be seen that the origin of this moiety can definitely be localized between 3-deoxy-D-arabinoheptulosonic acid-7-phosphate and shikimate.