10534-44-8

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The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products. Copyright

A convenient synthesis of dehydroquinic acid and its corresponding methyl ester are described. Protection of the trans diol of quinic acid, followed by PCC oxidation, gave fully protected dehydroquinic acid. This gave methyl dehydroquinate on mild acid ca

3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline qui

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Microcalorimetry and high-performance liquid chromatography (h.p.l.c.) have been used to conduct a thermodynamic investigation of reactions catalyzed by 3-dehydroquinate synthase and by 3-dehydroquinate dehydratase. These are the second and third reactions in the metabolic pathway leading to the formation of chorismate. The two reactions are: {DAHP(aq) = 3-dehydroquinate(aq) + phosphate(aq)} and {3-dehydroquinate(aq) = 3-dehydroshikimate(aq) + H2O(l)}. The h.p.l.c. measurements showed that the first reaction proceeded to completion and that the value of the apparent equilibrium constant for the second reaction was K′ = (4.6 ± 1.5) (Hepes buffer, temperature T = 298.15 K, pH = 7.50, and ionic strength Im = 0.065 mol·kg-1). Calorimetric measurements led to a molar enthalpy of reaction ΔrHm (cal) = -(50.9 ± 1.1) kJ·mol-1 (Hepes buffer, T = 298.15 K, pH = 7.46, Im = 0.070 mol·kg-1) for the first reaction and to ΔrHm (cal) = (2.3 ± 2.3) kJ·mol-1 (Hepes buffer, T = 298.15 K, pH = 7.42, Im = 0.069 mol·kg-1) for the second reaction. These results were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at T = 298.15 K and Im = 0 for chemical reference reactions involving specific ionic forms. For the reaction DAHP3-(aq) = 3-dehydroquinate-(aq) + HPO42-(aq), the standard molar enthalpy of reaction ΔrHmo = -(51.1 ± 4.5) kJ·mol-1. For the reaction 3-de-hydroquinate(aq) = (3-dehydroshikimate(aq) + H2O(l), the equilibrium constant K = (4.6 ± 1.5) and ΔrHmo = (2.3 ± 2.3) kJ·mol-1. A Benson type approach was used to estimate the standard molar entropy change ΔrSmo for the first reference reaction and led to the value K ≈ 2·1014 for this reaction. Values of the apparent equilibrium constants and the standard transformed Gibbs free energy changes ΔrGmo under approximately physiological conditions are given for the biochemical reactions.

Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292

3-Dehydroshikimate was formed with a yield of 57-77% from quinate via 3-dehydroquinate by two successive enzyme reactions, quinoprotein quinate dehydrogenase (QDH) and 3-dehydroquinate dehydratase, in the cytoplasmic membranes of acetic acid bacteria. 3-Dehydroshikimate was then reduced to shikimate (SKA) with NADP-dependent SKA dehydrogenase (SKDH) from the same organism. When SKDH was coupled with NADP-dependent D-glucose dehydrogenase (GDH) in the presence of excess D-glucose as an NADPH regenerating system, SKDH continued to produce SKA until 3-dehydroshikimate added initially in the reaction mixture was completely converted to SKA. Based on the data presented, a strategy for high SKA production was proposed.

A number of routes to 3-hydroxyiminodehydroquinate 4, one of the most potent inhibitors of type II dehydroquinase that is currently known, have been investigated. Methods based on the existing literature synthesis, i.e. oxime formation of a suitably C-4 a

Methyl 1,4,5-tris(trimethylsilyl)-3-dehydroquinate was synthesized in a three-step procedure and condensed with various nucleophiles (-CH2COOEt, -CH2SO2Ph, -CH2-CCH, H-).The diastereoselectivity of the reaction, leading to the creation of a new quaternary (or tetiary) asymmetric carbon atom was examined.The preferential axial attack on the C=O group of the cyclohexanone system is discussed.The deprotected products were evaluated as DHQase inhibitors. - Keywords: methyl 3-dehydroquinate; nucleophilic addition; diastereoselectivity.