3-Dehydroquinate synthase in germinating Phaseolus mungo seedlings.

Dehydroquinate synthase, an enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, was detected in cell-free extracts of etiolated Phaseolus mungo seedlings. The reaction product, 3-dehydroquinate, formed from [1-14C]DAHP was identified by paper-radiochromatography. The enzyme required NAD+ and Co2+ for activity.     

The shikimate pathway. III. 3-dehydroquinate synthetase of E. coli. Mechanistic studies by kinetic isotope effect.

The conversion of 3-deoxy D-arabino heptulosonate 7-phosphate to 3-dehydroquinate by the 3-dehydroquinate synthetase from E. coli is characterized by a low but significant kinetic isotope effect for tritium carried in position-5 of DAHP, while no isotope effect was detectable for tritium in position-4. This effect was observed at different pH nad is interpreted as a result of theintermediary of a 5-ketonic form of the substrate, formed in a preliminary non limiting step during the enzymic cyclization reaction. A tentative scheme for the 3-DHQ synthetase reaction is proposed involving five steps: oxidation by NAD+ in position-5, phsophate elimination after enolization, reduction with precedently formed NADH and cyclization by attack of the 2-carbonyl by the C-7 methylene group.     

Conversion of quinate to 3-dehydroshikimate by Ca-alginate-immobilized membrane of Gluconobacter oxydans IFO 3244 and subsequent asymmetric reduction of 3-dehydroshikimate to shikimate by immobilized cytoplasmic NADP-shikimate dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate:NADP+ oxidoreductase in Phaseolus mungo seedlings

Two associated enzymes, 3-dehydroquinate hydro-lyase (EC 4.2.1.10) and shikimate:NADP+ oxidoreductase (EC 1.1.1.25), have been purified from Phaseolus mungo seedlings. These enzymes were purified 6900- and 9700-fold, respectively, but they were not separable. Moreover, two activity bands of the shikimate:NADP+ oxidoreductase were detected after polyacrylamide gel electrophoresis and the two peaks also have 3-dehydroquinate hydro-lyase activity. The two forms of the associated enzymes showed only small differences in molecular weight, Km value, pH optimum and the responses to some inhibitors.     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

3-dehydroquinate production by oxidative fermentation and further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway

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 quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.     

5-Dehydro-3-deoxy-D-arabino-heptulosonic acid 7-phosphate. An intermediate in the 3-dehydroquinate synthase reaction.

3-Dehydroquinate synthase was purified to homogeneity from Escherichia coli. It was found to be a single polypeptide chain of Mr = approximately 57,000. Reaction mixtures of pure enzyme and the substrate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, were incubated for short times and treated with NaB3H4. The resulting 3-deoxyheptonic acid 7-phosphate was degraded with sodium periodate, and formic acid representing C-5 of the substrate was isolated. The presence of 3H in the formate corresponding to 15% of the enzyme was interpreted as indicating a 5-dehydro derivative of the substrate as an intermediate of the reaction. Quinic acid, resulting from reduction of 3-dehydroquinate with NaB3H4, was also isolated and degraded with periodate. The formate from C-4 of the quinate was unlabeled, indicating that 3,4-bisdehydroquinate is not an intermediate.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

Purification and Characterization of Membrane-Bound 3-Dehydroshikimate Dehydratase from Gluconobacter oxydans IFO 3244, A New Enzyme Catalyzing Extracellular Protocatechuate Formation

3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6–8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.     

The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose

The putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose was identified in the producer Actinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-D-glucose 4,6-dehydratase, acbB. The two flanking genes were acbA (dTDP-D-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299-302).     

Ultrastructural localization of coenzyme A phosphatase (CoA-Pase) activity to the GERL system in secretory ameloblasts of the rat incisor

Enzymatic hydrolysis of the monoester phosphate group from coenzyme A (CoA) was studied in rat incisor ameloblasts by incubating specimens from glutaraldehyde-fixed teeth in a cytochemical medium prepared with acetyl-CoA as substrate and lead ions as capture agent for phosphate. Ameloblasts incubated for 1 hr at 37 degrees C and at pH 5.0 in this medium showed reaction product localized almost exclusively along the trans (mature) aspect of the Golgi apparatus within a network of small granules and interconnecting tubular channels that comprise the GERL system in this cell. Reaction product was otherwise seen in trace amounts only within some Golgi saccules, a few lysosomal dense bodies and, in rare instances, within an occasional focal area of the endoplasmic reticulum. No selective staining of the GERL system was seen in control ameloblasts incubated at either pH 7.2 or pH 9.0 with acetyl-CoA as substrate, or incubated at pH 5.0 with dephospho-CoA as substrate. Control experiments at pH 5.0 also revealed that reaction product selectively stained the GERL system in ameloblasts when other molecules resembling CoA were used as substrate (e.g., crotonyl-CoA, 3'-NADP+), but not when adenosine 3'-monophosphate (3'-AMP) was used as substrate. That is, ameloblasts incubated at pH 5.0 with 3'-AMP showed heavy deposits of reaction product at many sites throughout the cell, including most lysosomal dense bodies, the Golgi saccules, the GERL system, most secretory granules, the nucleus, and extensively throughout the endoplasmic reticulum. These findings suggest that the GERL system of ameloblasts contains a CoA-specific phosphatase activity that may function to convert CoA to dephospho-CoA at acid pH. Biochemical studies included with this investigation further indicate that CoA-Pase activity saturates at exceptionally low concentrations of substrate (KM = 30 microM CoA) compared to other acid-dependent phosphatases.     

The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+-dependent.

We have demonstrated the co-purification in constant ratio of all five activities of the pentafunctional arom enzyme complex from Neurospora crassa. Progressive inactivation of the 3-dehydroquinate synthase component of the purified enzyme complex by chelating agents was blocked by the substrate, 3-deoxy-D-arabino-heptulosonate 7-phosphate, but not by the cofactor NAD+. Full activity was restored at Zn2+ concentrations as low as 0.05 nM. Atomic absorption data indicated that the intact enzyme complex contained 1 atom per subunit of tightly bound zinc. The arom 3-dehydroquinate synthase had a calculated turnover number of 19s-1, this being within the narrow range of values obtained for the other four activities of the intact multifunctional enzyme. The Km for 3-deoxy-D-arabino-heptulosonate 7-phosphate was 1.4 microM in a phosphate-free buffer; inorganic phosphate was a competitive inhibitor with respect to 3-deoxy-D-arabino-heptulosonate 7-phosphate.     

Monodeiodination of thyroxine to 3, 5, 3'-triiodothyronine and to 3, 3', 5'-triiodothyronine in isolated dog renal cortical tubuli

Monodeiodination of T4 to T3 and rT3 in the intact cells of dog renal tubuli and glomeruli was investigated. The tubuli and glomeruli were obtained by a sieve method. T4 (2 micrograms/ml) was incubated in Tris-HCl buffer, pH 7.5, with renal cells (180 micrograms protein/ml) and 5 mM DTT for 1 h at 37 degrees C and the T3 and rT3 generated during incubation were measured by specific radioimmunoassays. In order of decreasing activity, dog renal cortical tubuli, cortical homogenate, glomeruli and medullary tubuli were capable of converting T4 to T3. Net rT3 production from T4 in cortical tubuli was also greater than that in cortical homogenate. The conversion of T4 to T3 and also to rT3 in cortical tubuli was enzymatic in nature, since the reactions showed dependence on time and protein concentration; instability to heating; temperature and pH optimum. The production of T3 and rT3 from T4 was maximum at pH 6.5 and at pH 9.5, respectively, indicating that two different enzymic systems, a 5- and a 5'-monodeiodinase, might be involved in the deiodination of the tyrosyl and the phenolic ring of T4 in dog kidney.     

A novel 3-dehydroquinate dehydratase catalyzing extracellular formation of 3-dehydroshikimate by oxidative fermentation of Gluconobacter oxydans IFO 3244

In addition to the cytoplasmic soluble form of 3-dehydroquinate dehydratase (sDQD) (EC 4.1.2.10), a novel form of DQD occurring in the periplasmic space was found in Gluconobacter oxydans IFO 3244. The novel DQD, tentatively designated as pDQD, appeared to have a practical function involved in oxidative fermentation extracellularly coupling with membrane-bound quinoprotein quinate dehydrogenase (QDH) yielding 3-dehydroshikimate from quinate via 3-dehydroquinate. pDQD was not detached from the membrane by mechanical disruption or extraction with high salt, but was solubilized only with detergent. pDQD and sDQD were purified to homogeneity and compared as to their enzymatic properties. They showed the same apparent molecular weights and same catalytic properties, but they were distinct each other in subunit molecular mass, 16 kDa for pDQD and 47 kDa for sDQD.     

Purification and characterization of 3-dehydroquinate hydrolase and shikmate oxidoreductase. Evidence for a bifunctional enzyme

The basis for the physical association of 3-dehydroquinate dehydratase (3-dehydroquinate hydrolyase, EC 4.2.1.10) and shikimate dehydrogenase (shikimate: NADP+ 3-oxidoreductase, EC 1.1.1.25) in higher plants was investigated. The enzymes were extracted from the moss Physcomitrella patens and were purified to homogeneity. Determinations of subunit sizes were made by sodium dodecyl sulfate gel electrophoresis and gel exclusion chromatography in 6 M guanidinium chloride. Results from these studies demonstrate that both enzyme activities are carried out by a single polypeptide.     

Schistosoma mansoni and Biomphalaria glabrata: Ultrastructural localization of enzymes with diaminobenzidine in larvae and host digestive glands.

All mitochondria contained reaction product when daughter sporocysts of Schistosoma mansoni and digestive glands of the snail host, Biomphalaria glabrata, were cytochemically incubated for 45 or 60 min with alkaline 3, 3′-diaminobenzidine (DAB) at pH 7.4 and 9.0. The pigment marked the presence of cytochrome c-cytochrome oxidase activity, and was not found in parasite or gland tissues incubated with DAB and KCN at pH 7.4, 9.0, and 9.8.After incubation for 45 min in the pH 7.4 DAB medium, tegumental mitochondria in young intrasporocyst cercariae showed DAB reaction product, but little or none of the pigment was found in tegumental mitochondria of older, glycocalyx-covered cercariae. In contrast, mitochondria of subtegumental cells were strongly DAB positive at all stages of intrasporocyst cercarial development. No differences in DAB reactivity were detected in mitochondria of sporocysts, or of infected and uninfected host gland cells.Reaction product was found in certain vacuoles of digestive cells incubated in the pH 9.8 DAB medium with KCN, but not in the pH 9.8 DAB medium with amino triazole, or in the pH 7.4 DAB medium. No peroxisomes or microperoxisomes were found in the tissues studied.     

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.     

The interaction of phosphonate and homophosphonate analogues of 3-deoxy-D-arabino heptulosonate 7-phosphate with 3-dehydroquinate synthetase from Escherichia coli

Phosphonate and homophosphonate analogues of 3-deoxy-D-$\text{arabino}$ heptulosonate 7-phosphate and D-$\text{gluco}$ heptulosonate 7-phosphate behave as competitive inhibitors of 3-dehydroquinate synthetase. Phosphonates have better affinities than homophosphonates and protect efficiently the enzyme against thermal denaturation. No evidence has been obtained for 5-keto phosphonate intermediate formation in the interaction of such analogues with 3-dehydroquinate synthetase and NAD⁺.