Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.

Science.gov (United States)

Chavarría, Max; Durante-Rodríguez, Gonzalo; Krell, Tino; Santiago, César; Brezovsky, Jan; Damborsky, Jiri; de Lorenzo, Víctor

2014-01-01

Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (Cra(PP)) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between Cra(PP) and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra(PP) represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of Cra(PP) for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

Dinuclear copper(II) complexes with {Cu2(mu-hydroxo)bis(mu-carboxylato)}+ cores and their reactions with sugar phosphate esters: A substrate binding model of fructose-1,6-bisphosphatase.

Science.gov (United States)

Kato, Merii; Tanase, Tomoaki; Mikuriya, Masahiro

2006-04-03

-ray crystallography to involve two different tetranuclear complexes with alpha- and beta-anomers of D-Fru-1,6-P2, [Cu4(mu-alpha-D-Fru-1,6-P2)(XDK)2(phen)4] and [Cu4(mu-beta-D-Fru-1,6-P2)(XDK)2(phen)4], in which the D-Fru-1,6-P2 tetravalent anion bridges the two [Cu2(XDK)(phen)2]2+ units through the C1 and C6 phosphate groups in a mu-1,3-O,O' bidentate fashion (Cu...Cu = 4.042(2)-4.100(2) A). Notably, the structure with alpha-D-Fru-1,6-P2 demonstrated the presence of a strong hydrogen bond between the C2 hydroxyl group and the C1 phosphate oxygen atom, which may support the previously proposed catalytic mechanism in the active site of fructose-1,6-bisphosphatase.

Diagnosis of Hunter's syndrome carriers; radioactive sulphate incorporation into fibroblasts in the presence of fructose 1-phosphate

International Nuclear Information System (INIS)

Toennesen, T.; Lykkelund, C.; Guettler, F.

1982-01-01

Mutual correction of co-cultivated fibroblasts from patients with Hunter's and Hurler's syndrome could be inhibited by either fructose 1-phosphate or mannose 6-phosphate. In the presence of fructose 1-phosphate a 50% mixture of fibroblasts from a patient with Hunter's syndrome and a normal homozygous individual showed an increased 35 S-sulphate incorporation into acid mucopolysaccharides. When fibroblast cultures from one obligate and two possible carriers of Hunter's syndrome were tested for 35 S-sulphate incorporation, the cultures showed either twice the normal 35 S-sulphate incorporation into acid mucopolysaccharides in the presence of fructose 1-phosphate or an abnormally high incorporation in the presence as well as in the absence of the sugar phosphate. (orig.)

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate

International Nuclear Information System (INIS)

Gardberg, Anna; Abendroth, Jan; Bhandari, Janhavi; Sankaran, Banumathi; Staker, Bart

2011-01-01

While other aldolases crystallize readily in the apo form, diffraction-quality crystals of B. henselae aldolase could only be obtained in the presence of the native substrate. The quaternary structure is tetrameric, as is typical of aldolases. Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 72.39, b = 127.71, c = 157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site

Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase.

Science.gov (United States)

Roldán, Raquel; Sanchez-Moreno, Israel; Scheidt, Thomas; Hélaine, Virgil; Lemaire, Marielle; Parella, Teodor; Clapés, Pere; Fessner, Wolf-Dieter; Guérard-Hélaine, Christine

2017-04-11

d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new radiochemical assay for fructose-1,6-diphosphatase in human leucocytes

International Nuclear Information System (INIS)

Janssen, A.J.M.; Trijbels, F.J.M.

1982-01-01

Fructose-1,6-diphosphatase (D-fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11, FDPase) is one of the key enzymes of the gluconeogenic pathway. Measuring the activity both in the presence and in the absence of AMP yields the true FDPase activity, corrected for non-specific phosphatase activity. In this paper the authors introduce a new radiochemical assay for FDPase, based on the decarboxylating activity of 6-phosphogluconate dehydrogenase. One molecule [U- 14 C]fructose-1,6-diphosphate yields one molecule 14 CO 2 which can be captured in strongly basic solutions and counted in a liquid scintillation counter. (Auth.)

Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases.

Science.gov (United States)

Lee, Seon-Hwa; Hong, Seung-Hye; An, Jung-Ung; Kim, Kyoung-Rok; Kim, Dong-Eun; Kang, Lin-Woo; Oh, Deok-Kun

2017-05-16

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II D-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of D-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and L-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases L-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.

Complex formation of uranium(VI) with fructose and glucose phosphates

International Nuclear Information System (INIS)

Koban, A.; Geipel, G.; Bernhard, G.; Fanghaenel, T.

2002-01-01

The uptake of heavy metals into plants is commonly quantified by the soil-plant transfer factor. Up to now little is known about the chemical speciation of actinides in plants. To compare the obtained spectroscopic data of uranium complexes in plants with model compounds, we investigate the complexation of uranium with relevant bioligands of various functionalities. A very important class of ligands consists of phosphate esters, which serve as phosphate group and energy transmitters as well as energy storage media in biological systems. Heavy metal ions bound to the phosphate esters can be transported into living cells and then deposited. Therefore, in our study we present the results of uranium complexation with glucose-6-phosphate (G6P), and fructose-6-phosphate (F6P) obtained by time-resolved laser-induced fluorescence spectroscopy (TRLFS). The experiments were performed at a fixed uranyl concentration (10 -5 M) as a function of the ligand concentrations (10 -5 to 10 -3 M) in a pH range from 2 to 4.5. For the glucose phosphate system we observed, using increasing ligand concentrations, a decrease in the fluorescence intensity and a small red shift of the emission bands. From this we conclude that the complexed uranyl glucose phosphate species show only minor or no fluorescence properties. The TRLFS spectra of the glucose phosphate samples indicated the presence of a single species with fluorescence properties. This species has a lifetime of approximately 1.5 μs and was identified as the free uranyl ion. An opposite phenomenon was observed for the fructose phosphate system: there was no decrease in fluorescence intensity. However, a strong red shift of the spectra was observed, illustrating the fluorescence properties of the uranyl fructose phosphate complex. The TRLFS spectra of the fructose phosphate system showed a second lifetime ( 2 2+ UO 2 (lig) x (2-y)+ + y H + (lig = sugar phosphate). Applying the mass action law and transformation to the logarithmic

Rv2131c gene product: An unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase

International Nuclear Information System (INIS)

Gu Xiaoling; Chen Mao; Shen Hongbo; Jiang Xin; Huang Yishu; Wang Honghai

2006-01-01

Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from Mycobacterium tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1,6-bisphosphatase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with K m of 0.22 ± 0.03 mM for inositol-1-phosphate and K m of 0.45 ± 0.05 mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1,6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was 12-fold more resistant to inhibition by lithium (IC 5 ∼ 60 mM). Based on the substrate specificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1,6-bisphosphatase (FBPase IV)

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

Science.gov (United States)

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

Science.gov (United States)

Messner, Christoph B; Driscoll, Paul C; Piedrafita, Gabriel; De Volder, Michael F L; Ralser, Markus

2017-07-11

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Pegan, Scott D. [Univ. of Denver, CO (United States); Rukseree, Kamolchanok [National Center for Genetic Engineering and Biotechnology (BIOTEC), Tha Khlong (Thailand); Capodagli, Glenn C. [Univ. of Denver, CO (United States); Baker, Erica A. [Univ. of Denver, CO (United States); Krasnykh, Olga [Univ. of Illinois, Chicago, IL (United States); Franzblau, Scott G. [Univ. of Illinois, Chicago, IL (United States); Mesecar, Andrew D. [Purdue Univ., West Lafayette, IN (United States)

2013-01-08

The class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprises one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Furthermore, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

Determination of fructose metabolic pathways in normal and fructose-intolerant children: A 13C NMR study using [U-13C]fructose

International Nuclear Information System (INIS)

Gopher, A.; Lapidot, A.; Vaisman, N.; Mandel, H.

1990-01-01

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U- 13 C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13 C NMR spectra of plasma glucose. Significantly lower values (∼3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13 C NMR measurement of plasma [ 13 C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13 C atoms at glucose C-4 ( 13 C 3 - 13 C 4 - 13 C 5 ) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only ∼50% of the total amount of hepatic fructose conversion to glucose. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [ 13 C]glucose formation from a trace amount of [U- 13 C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism

Influence of fructose on the diffusion of potassium hydrogen phosphate in aqueous solutions at 25 °C

International Nuclear Information System (INIS)

Verissimo, Luis M.P.; Teigão, Joana M.M.; Ramos, M. Luísa; Burrows, Hugh D.; Esteso, Miguel A.; Ribeiro, Ana C.F.

2016-01-01

Highlights: • Diffusion coefficients of aqueous systems of fructose and potassium hydrogen phosphate measured with Lobo’s cell. • Influence of the fructose on the diffusion of potassium hydrogen phosphate. • Interactions between of hydrogen phosphate anion and fructose. - Abstract: Diffusion coefficients have been measured at 25 °C for potassium hydrogen phosphate (K_2HPO_4, 0.101 mol kg"−"1) in aqueous solutions containing various concentrations of fructose from (0.001 to 0.101) mol kg"−"1, using a conductimetric cell (the Lobo cell) coupled to an automatic data acquisition system. Significant effects of fructose were observed on the diffusion of K_2HPO_4 in these mixtures, which are attributed to the interaction between HPO_4"2"− anion (or other protonated forms) and fructose. Support for this comes from "1H and "1"3C NMR spectroscopy, which are compatible with binding between the anomeric forms of D-fructose and the HPO_4"2"− anion.

Regulation of dual glycolytic pathways for fructose metabolism in heterofermentative Lactobacillus panis PM1.

Science.gov (United States)

Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji

2013-12-01

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli that use the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway as their central metabolic pathway and are reportedly unable to grow on fructose as a sole carbon source. We isolated a variant PM1 strain capable of sporadic growth on fructose medium and observed its distinctive characteristics of fructose metabolism. The end product pattern was different from what is expected in typical group III lactobacilli using the 6-PG/PK pathway (i.e., more lactate, less acetate, and no mannitol). In addition, in silico analysis revealed the presence of genes encoding most of critical enzymes in the Embden-Meyerhof (EM) pathway. These observations indicated that fructose was metabolized via two pathways. Fructose metabolism in the PM1 strain was influenced by the activities of two enzymes, triosephosphate isomerase (TPI) and glucose 6-phosphate isomerase (PGI). A lack of TPI resulted in the intracellular accumulation of dihydroxyacetone phosphate (DHAP) in PM1, the toxicity of which caused early growth cessation during fructose fermentation. The activity of PGI was enhanced by the presence of glyceraldehyde 3-phosphate (GAP), which allowed additional fructose to enter into the 6-PG/PK pathway to avoid toxicity by DHAP. Exogenous TPI gene expression shifted fructose metabolism from heterolactic to homolactic fermentation, indicating that TPI enabled the PM1 strain to mainly use the EM pathway for fructose fermentation. These findings clearly demonstrate that the balance in the accumulation of GAP and DHAP determines the fate of fructose metabolism and the activity of TPI plays a critical role during fructose fermentation via the EM pathway in L. panis PM1.

Engineering the donor selectivity of D-fructose-6-phosphate aldolase for biocatalytic asymmetric cross-aldol additions of glycolaldehyde.

Science.gov (United States)

Szekrenyi, Anna; Soler, Anna; Garrabou, Xavier; Guérard-Hélaine, Christine; Parella, Teodor; Joglar, Jesús; Lemaire, Marielle; Bujons, Jordi; Clapés, Pere

2014-09-22

D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

Energy Technology Data Exchange (ETDEWEB)

Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

2008-01-01

Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

Cra regulates the cross-talk between the two branches of the phosphoenolpyruvate : phosphotransferase system of Pseudomonas putida.

Science.gov (United States)

Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

2013-01-01

The gene that encodes the catabolite repressor/activator, Cra (FruR), of Pseudomonas putida is divergent from the fruBKA operon for the uptake of fructose via the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS(Fru)). The expression of the fru cluster has been studied in cells growing on substrates that change the intracellular concentrations of fructose-1-P (F1P), the principal metabolic intermediate that counteracts the DNA-binding ability of Cra on an upstream operator. While the levels of the regulator were not affected by any of the growth conditions tested, the transcription of fruB was stimulated by fructose but not by the gluconeogenic substrate, succinate. The analysis of the P(fruB) promoter activity in a strain lacking the Cra protein and the determination of key metabolites revealed that this regulator represses the expression of PTS(Fru) in a fashion that is dependent on the endogenous concentrations of F1P. Because FruB (i.e. the EI-HPr-EIIA(Fru) polyprotein) can deliver a high-energy phosphate to the EIIA(Ntr) (PtsN) enzyme of the PTS(Ntr) branch, the cross-talk between the two phosphotransferase systems was examined under metabolic regimes that allowed for the high or low transcription of the fruBKA operon. While fructose caused cross-talk, succinate prevented it almost completely. Furthermore, PtsN phosphorylation by FruB occurred in a Δcra mutant regardless of growth conditions. These results traced the occurrence of the cross-talk to intracellular pools of Cra effectors, in particular F1P. The Cra/F1P duo seems to not only control the expression of the PTS(Fru) but also checks the activity of the PTS(Ntr) in vivo. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

1,5-Anhydro-D-fructose: regioselective acylation with fatty acids

DEFF Research Database (Denmark)

Lundt, Inge; Andersen, Søren Møller; Marcussen, Jan

1999-01-01

Regioselective acylation of 1,5-anhydro-D-fructose was performed with dodecanoic acid to give 1,5-anhydro-6-O-dodecanoyl-D-fructose, chemically in 50% yield and enzymatically in quantitative yield. Quantitative conversions were also obtained using hexadecanoic and octadecanoic acids as acyl donors...

Fructose 1-phosphate is the preferred effector of the metabolic regulator Cra of Pseudomonas putida.

Science.gov (United States)

Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M; de Lorenzo, Víctor

2011-03-18

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5'-TTAAACGTTTCA-3' (K(D) = 26.3 ± 3.1 nM) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a K(D) of 209 ± 20 nM. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.

Competitive binding assay for fructose 2,6-bisphosphate

International Nuclear Information System (INIS)

Thomas, H.; Uyeda, K.

1986-01-01

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P 2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol promoter) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6[2- 32 P]P 2 and samples containing varying concentrations of fructose 2,6-P 2 . The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P 2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P 2 , ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. The new assay is simple, direct, rapid, and does not require pretreatment

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Science.gov (United States)

Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

2014-08-01

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

STRUCTURAL INSIGHTS INTO SUBSTRATE BINDING AND STEREOSELECTIVITY OF GIARDIA FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE*

Science.gov (United States)

Galkin, Andrey; Li, Zhimin; Li, Ling; Kulakova, Liudmila; Pal, Lipika R.; Dunaway-Mariano, Debra; Herzberg, Osnat

2009-01-01

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA)1 is a member of the Class II zinc-dependent aldolase family that catalyzes the cleavage of D-fructose-1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced by an alanine residue renders the enzyme inactive. A comparison of the crystal structures of the D83A FBPA in complex with FBP and of the wild-type FBPA in the unbound state revealed a substrate induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn2+. Upon FBP binding, the Zn2+ shifts up to 4.6 Å towards the catalytic Asp83, which brings the metal within coordination distance to the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor D-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analog of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA/FBP structure. The mechanism invokes coordination and co-planarity of the Zn2+ with the FBP’s O-C(3)-C(4)-O concomitant with coordination of Asp83 carboxylic group. Catalysis is accompanied by movement of Zn2+ to a site co-planar with the O-C(2)-C(3)-O of the DHAP. glFBPA exhibit strict substrate specificity towards FBP and

Effects of oral D-tagatose, a stereoisomer of D-fructose, on liver metabolism in man as examined by 31P-magnetic resonance spectroscopy.

Science.gov (United States)

Buemann, B; Gesmar, H; Astrup, A; Quistorff, B

2000-10-01

D-tagatose, which is a stereoisomer of D-fructose, is phosphorylated to D-tagatose-1-phosphate by fructokinase in the liver. Because of a slow degradation rate of D-tagatose-1-phosphate, this substance may accumulate, and ingested D-tagatose may therefore cause a longer lasting reduction in inorganic phosphate (Pi) and adenosine triphosphate (ATP) levels in the liver compared with D-fructose. Similar to what is seen in patients with hereditary fructose intolerance, this may increase purine nucleotide degradation and thereby increase uric acid production. The effect of 30 g D-tagatose or D-fructose administered orally on ketohexose-1-phosphates, ATP, and Pi levels in the liver was studied by 31P-magnetic resonance spectroscopy (PMRS) in 5 young male volunteers. Blood and urine were collected to detect a possible increased uric acid production. A peak at 5.2 ppm assigned as D-tagatose-1-phosphate equivalent to about 1 mmol/L was found in the spectrum within 30 minutes after D-tagatose was administered in all subjects. Concomitantly, ATP was reduced by about 12% (P effects had vanished after 150 minutes. Serum uric acid concentration was increased by 17% 50 minutes after D-tagatose (P effect of D-tagatose. No changes in 31PMRS spectra or serum uric acid concentration were found after D-fructose. These results suggest that a moderate intake of D-tagatose may affect liver metabolism by phosphate trapping despite the fact that the sugar may only be incompletely absorbed in the gut.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

Science.gov (United States)

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Fructose 2,6-bisphosphate and its phosphorothioate analogue. Comparison of their hydrolysis and action on glycolytic and gluconeogenic enzymes.

OpenAIRE

Rider, M H; Kuntz, D A; Hue, L

1988-01-01

Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofru...

Synthesis of 4-O-glycosylated 1,5-anhydro-D-fructose and of 1,5-anhydro-D-tagatose from a common intermediate 2,3-O-isopropylidene -D-fructose

DEFF Research Database (Denmark)

Agoston, Karoly; Dekany, Gyula; Lundt, Inge

2009-01-01

Four novel disaccharides of glycosylated 1,5-anhydro-D-ketoses have been prepared: 1,5-anhydro-4-O-b-D-glucopyranosyl-D-fructose, 1,5-anhydro-4-O-b-D-galactopyranosyl-D-fructose, 1,5-anhydro-4-O-b-D-glucopyranosyl-D-tagatose and 1,5-anhydro-4-O-b-D-galactopyranosyl-D-tagatose. The common...... intermediate, 1,5-anhydro-2,3-O-isopropylidene-b-D-fructopyranose, was prepared from D-fructose and converted into the D-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The prepared anhydro-ketoses were glycosylated and deprotected to the disaccharides....

Synthesis of 4-O-glycosylated 1,5-anhydro-D-fructose and of 1,5-anhydro-D-tagatose from a common intermediate 2,3-O-isopropylidene-D-fructose.

Science.gov (United States)

Agoston, Károly; Dékány, Gyula; Lundt, Inge

2009-05-26

Four novel disaccharides of glycosylated 1,5-anhydro-D-ketoses have been prepared: 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-tagatose, and 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-beta-D-fructopyranose, was prepared from D-fructose and was converted into the D-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.

Crystal structure of β-d,l-fructose

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Tomohiko Ishii

2015-10-01

Full Text Available The title compound, C6H12O6, was crystallized from an aqueous solution of equimolar mixture of d- and l-fructose (1,3,4,5,6-pentahydroxyhexan-2-one, arabino-hexulose or levulose, and it was confirmed that d-fructose (or l-fructose formed β-pyranose with a 2C5 (or 5C2 conformation. In the crystal, two O—H...O hydrogen bonds between the hydroxy groups at the C-1 and C-3 positions, and at the C-4 and C-5 positions connect homochiral molecules into a column along the a axis. The columns are linked by other O—H...O hydrogen bonds between d- and l-fructose molecules, forming a three-dimensional network.

Genetic Evidence for the Physiological Significance of the d-Tagatose 6-Phosphate Pathway of Lactose and d-Galactose Degradation in Staphylococcus aureus1

Science.gov (United States)

Bissett, Donald L.; Anderson, Richard L.

1974-01-01

Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain. PMID:4277494

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition.

Science.gov (United States)

Wolf, Nina M; Gutka, Hiten J; Movahedzadeh, Farahnaz; Abad-Zapatero, Celerino

2018-04-01

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

A new chemical synthesis of Ascopyrone P from 1,5-anhydro-D-fructose

DEFF Research Database (Denmark)

Andreassen, Mikkel; Lundt, Inge

2006-01-01

The naturally occurring antioxidant Ascopyrone P (1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose, 1) was prepared from the rare sugar 1,5-anhydro-D-fructose (AF, 3) in three steps in an overall yield of 36%. Thus, acetylation of 3 afforded the enolone 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero......The naturally occurring antioxidant Ascopyrone P (1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose, 1) was prepared from the rare sugar 1,5-anhydro-D-fructose (AF, 3) in three steps in an overall yield of 36%. Thus, acetylation of 3 afforded the enolone 3,6-di-O-acetyl-1,5-anhydro-4-deoxy...

Crystallization and preliminary X-ray characterization of the glpX-encoded class II fructose-1,6-bisphosphatase from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Gutka, Hiten J.; Franzblau, Scott G.; Movahedzadeh, Farahnaz; Abad-Zapatero, Cele

2011-01-01

The crystallization and preliminary X-ray crystallographic analysis of the glpX-encoded class II fructose-1,6-bisphosphatase from M. tuberculosis in the apo form is reported. Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7 Å and belonged to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 131.3, c = 143.2 Å. The structure has been solved by molecular replacement and is currently undergoing refinement

Preliminary X-ray crystallographic analysis of the d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis

International Nuclear Information System (INIS)

Petrareanu, Georgiana; Balasu, Mihaela C.; Zander, Ulrich; Scheidig, Axel J.; Szedlacsek, Stefan E.

2010-01-01

The expression, purification, preliminary crystallization and crystallographic analysis of phosphoketolase from L. lactis ssp. lactis (strain IL 1403) are reported. Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon–carbon bond of the specific substrate d-xylulose 5-phosphate (or d-fructose 6-phosphate) to give acetyl phosphate and d-glyceraldehyde 3-phosphate (or d-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P2 1 . Diffraction data were obtained to a resolution of 2.2 Å

Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

Science.gov (United States)

Lee, Jung-Kul; Pan, Cheol-Ho

2013-01-01

D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

Directory of Open Access Journals (Sweden)

Woo-Suk Jung

Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes.

Science.gov (United States)

Ciudad, C J; Massagué, J; Salavert, A; Guinovart, J J

1980-03-20

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.

Molecular and biochemical characterizations of three fructose-1,6-bisphosphate aldolases from Clonorchis sinensis.

Science.gov (United States)

Li, Shan; Bian, Meng; Wang, Xiaoyun; Chen, Xueqing; Xie, Zhizhi; Sun, Hengchang; Jia, Feifei; Liang, Pei; Zhou, Chenhui; He, Lei; Mao, Qiang; Huang, Bo; Liang, Chi; Wu, Zhongdao; Li, Xuerong; Xu, Jin; Huang, Yan; Yu, Xinbing

2014-01-01

Fructose-1,6-bisphosphate aldolase (FbA) is a ubiquitous enzyme in glycolysis. In the present study, we screened out three distinct genes encoding FbA isozymes (CsFbAs, CsFbA-1/2/3) from Clonorchis sinensis (C. sinensis) and characterized their sequences and structures profiles as well as biochemical properties. The amino acid sequences of CsFbAs shared homology with those of Class I FbAs from other species. The putative quaternary structures revealed that CsFbA-2 and CsFbA-3 were tetramers, while CsFbA-1 was dimer. Recombinant CsFbA-2 and CsFbA-3 (rCsFbA-2/3) were confirmed to be Class I FbAs for their stable enzymatic activities in the presence of EDTA or metal ions. However, recombinant CsFbA-1 (rCsFbA-1) did not show the catalytic activity, which might be due to the inappropriate fold and interaction between its subunits. Both rCsFbA-2 and rCsFbA-3 showed similar enzymatic properties such as optimal temperatures and broad pH ranges that similar to human FbA isozymes. They showed relatively higher affinities for fructose-1,6-bisphosphate (FBP) than fructose-1-phosphate (F-1-P). Their kcat ratios of FBP to F-1-P were in accordance with those of human FbA-A or C. In addition, CsFbAs were differentially transcribed in the developmental stages of C. sinensis, suggesting their essential roles throughout the life stages. Extensive distribution of CsFbAs in adult worms indicated that ubiquitous activities of CsFbAs took place in these organs. Collectively, these results suggested that long-term parasitic environment might adapt these isozymes similar to host FbAs for metabolic requirement. Our study will provide new insight into CsFbAs in the glycometabolism of C. sinensis and relationship between the host and the parasite. Copyright © 2014 Elsevier B.V. All rights reserved.

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase

International Nuclear Information System (INIS)

Boucher, Lauren E.; Bosch, Jürgen

2014-01-01

The structure of T. gondii fructose-1,6-bisphosphate aldolase, a glycolytic enzyme and structural component of the invasion machinery, was determined to a resolution of 2.0 Å. The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-@@bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22 1 2 1 , with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented

Combined dehydration/(transfer)-hydrogenation of C6-sugars (D-glucose and D-fructose) to gamma-valerolactone using ruthenium catalysts

NARCIS (Netherlands)

Heeres, Hans; Handana, Ratna; Chunai, Dai; Rasrendra, Carolus Borromeus; Girisuta, Buana; Heeres, Hero Jan

2009-01-01

gamma-Valerolactone (GVL) is considered a very interesting green, bio-based platform chemical with high application potential. We here describe research activities on the one-pot catalytic synthesis of GVL from C6-sugar sources (D-glucose, D-fructose, sucrose and cellulose) using an acid catalyst in

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

Directory of Open Access Journals (Sweden)

Neal J Dawson

Full Text Available One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05. A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively. Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

Science.gov (United States)

Dawson, Neal J; Biggar, Kyle K; Storey, Kenneth B

2013-01-01

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

Energy Technology Data Exchange (ETDEWEB)

Koo, Hyun-Young [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States); Miyashita, Michio [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States); Department of Pediatrics, Nihon University School of Medicine, Itabashi, Tokyo (Japan); Simon Cho, B.H. [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States); Harlan E. Moore Heart Research Foundation, 503 South Sixth Street, Champaign, IL 61820 (United States); Nakamura, Manabu T., E-mail: mtnakamu@illinois.edu [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

2009-12-11

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9x higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

International Nuclear Information System (INIS)

Koo, Hyun-Young; Miyashita, Michio; Simon Cho, B.H.; Nakamura, Manabu T.

2009-01-01

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9x higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.

Antioxidant activity of the melanoidin fractions formed from D-Glucose and D-Fructose with L-Asparagine in the Maillard reaction

Directory of Open Access Journals (Sweden)

A.P Echavarría

2013-01-01

Full Text Available Melanoidins formed at the last stage of the Maillard reaction have been shown to possess certain functional properties, such as antioxidant activity. In order to gain more insight into these functional properties, soluble model systems melanoidins from L - Asparagine with D - glucose or D - fructose fractionating by ultrafiltration were analyzed. The fractionating/concentration sequence of the melanoidin fraction (1 - 300 kDa enabled five fractions to be produced. Additionally, the absorption of melanoidins was measured at different wavelengths (280, 325, 405 and browning at 420 nm. The fractionati on effect of melanoidin systems on the color intensity, UV - absorbance scan wavelengths (nm, CIE, L*, a*, b* parameters and antioxidant activity were measured. For this purpose, antioxidant activity was evaluated through the free radical scavenging activit y, including 1,1 - diphenyl - 2 - picryl - hydrazil (DPPH and 2,20 - azinobis (3 - ethylbenothiazoline - 6 - sulfonic acid, diammonium salt (ABTS. The results showed that the absorption of the melanoidins formed from Glucose/L - Asn was higher than for those derived from Fructose/L - Asn. On the other hand, their antioxidant power was lower than that for melanoidins formed from Fructose/L - Asn systems.

Structure of 1,5-Anhydro-D-Fructose: X-ray Analysis of Crystalline Acetylated Dimeric Forms

DEFF Research Database (Denmark)

Lundt, Inge; Andersen, Søren Møller; Marcussen, Jan

1998-01-01

Acetylation of 1,5-anhydro-D-fructose under acidic conditions gave two crystalline acetylated dimeric forms, which by X-ray analysis were shown to be diastereomeric spiroketals formed between C-2 and C-2´/C-3´. The structures of the compounds differed only at the configuration at C-2. Acetylation...... or benzoylation of 1,5-anhydro-D-fructose in pyridine yielded 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enos-2-ulopyra -nose or crystalline 1,5-anhydro-3,6-di-O-benzoyl-4-deoxy-D-glycero-hex-3-enos-2-ulo-py ranose....

Escherichia coli Phosphoenolpyruvate Dependent Phosphotransferase System. Copurification of HPr and α1-6 Glucan

NARCIS (Netherlands)

Dooijewaard, G.; Roossien, F.F.; Robillard, G.T.

1979-01-01

A rapid, high-yield procedure has been developed for the purification of HPr from the Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. During this procedure, the protein copurifies with a 2500-dalton homopolysaccharide which we have identified as α1-6 glucan. The results of

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

Energy Technology Data Exchange (ETDEWEB)

Gowda, Giri; Sagurthi, Someswar Rao [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

2008-02-01

The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida*

Science.gov (United States)

Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M.; de Lorenzo, Víctor

2011-01-01

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (KD = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a KD of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida. PMID:21239488

Genetic analysis of fructose-1,6-bisphosphatase (FBPase) deficiency in nine consanguineous Pakistani families.

Science.gov (United States)

Ijaz, Sadaqat; Zahoor, Muhammad Yasir; Imran, Muhammad; Ramzan, Khushnooda; Bhinder, Munir Ahmad; Shakeel, Hussain; Iqbal, Muhammad; Aslam, Asim; Shehzad, Wasim; Cheema, Huma Arshad; Rehman, Habib

2017-10-26

Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited metabolic disorder characterized by recurrent episodes of hypoglycemia, ketosis and lactic acidosis. FBPase is encoded by FBP1 gene and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate in the last step of gluconeogenesis. We report here FBP1 mutations in nine consanguineous Pakistani families affected with FBPase deficiency. Nine families having one or two individuals affected with FBPase deficiency were enrolled over a period of 3 years. All FBP1 exonic regions including splicing sites were PCR-amplified and sequenced bidirectionally. Familial cosegregation of mutations with disease was confirmed by direct sequencing and PCR-RFLP analysis. Three different FBP1 mutations were identified. Each of two previously reported mutations (c.472C>T (p.Arg158Trp) and c.841G>A (p.Glu281Lys)) was carried by four different families. The ninth family carried a novel 4-bp deletion (c.609_612delAAAA), which is predicted to result in frameshift (p.Lys204Argfs*72) and loss of FBPase function. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. FBPase deficiency is often fatal in the infancy and early childhood. Early diagnosis and prompt treatment is therefore crucial to preventing early mortality. We recommend the use of c.472C>T and c.841G>A mutations as first choice genetic markers for molecular diagnosis of FBPase deficiency in Pakistan.

Structure of a rabbit muscle fructose-1, 6-bisphosphate aldolase A dimer variant

Energy Technology Data Exchange (ETDEWEB)

Sherawat, Manashi [Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2394 (United States); Tolan, Dean R., E-mail: tolan@bu.edu [Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215 (United States); Allen, Karen N., E-mail: tolan@bu.edu [Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2394 (United States)

2008-05-01

The X-ray crystallographic structure of a dimer variant of fructose-1, 6-bisphosphate aldolase demonstrates a stable oligomer that mirrors half of the native tetramer. The presence of product demonstrates that this is an active form. Fructose-1, 6-bisphosphate aldolase (aldolase) is an essential enzyme in glycolysis and gluconeogenesis. In addition to this primary function, aldolase is also known to bind to a variety of other proteins, a property that may allow it to perform ‘moonlighting’ roles in the cell. Although monomeric and dimeric aldolases possess full catalytic activity, the enzyme occurs as an unusually stable tetramer, suggesting a possible link between the oligomeric state and these noncatalytic cellular roles. Here, the first high-resolution X-ray crystal structure of rabbit muscle D128V aldolase, a dimeric form of aldolase mimicking the clinically important D128G mutation in humans associated with hemolytic anemia, is presented. The structure of the dimer was determined to 1.7 Å resolution with the product DHAP bound in the active site. The turnover of substrate to produce the product ligand demonstrates the retention of catalytic activity by the dimeric aldolase. The D128V mutation causes aldolase to lose intermolecular contacts with the neighboring subunit at one of the two interfaces of the tetramer. The tertiary structure of the dimer does not significantly differ from the structure of half of the tetramer. Analytical ultracentrifugation confirms the occurrence of the enzyme as a dimer in solution. The highly stable structure of aldolase with an independent active site is consistent with a model in which aldolase has evolved as a multimeric scaffold to perform other noncatalytic functions.

Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances.

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Du Toit W P Schabort

Full Text Available The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production.

Structural Diversity Within the Mononuclear and Binuclear Active Sites of N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase

Energy Technology Data Exchange (ETDEWEB)

Hall,R.; Brown, S.; Fedorov, A.; Fedorov, E.; Xu, C.; Babbitt, P.; Almo, S.; Raushel, F.

2007-01-01

NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.

New Products from 1,5-Anhydro-D-fructose

DEFF Research Database (Denmark)

Andreassen, Mikkel; Lundt, Inge

1,5-Anhydro-D-fructose 1 (AF) is now available in larger amounts by enzymatic degradation of starch, and thus the utility of AF is the basis of the NEPSA project . A review covering the chemistry and biochemistry of AF has recently been published . In our ongoing research to use AF as a chiral...... investigation. EU-program : NEw Products from Starch derived Anhydro-D- fructose Andersen, S.; Lundt, I.; Marcussen, J.; Yu, S. Carb.Res. 2002, 337, 873-880 Andersen, S.; Isak, T.; Jensen, H. M.; Marcussen, J.; Yu, S. Anti-oxidant. PCT Int. Patent WO 0056838, 2000...

Design, synthesis and in vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

Energy Technology Data Exchange (ETDEWEB)

Dias, Danielle F.; Alves, Ricardo J., E-mail: ricardodylan@farmacia.ufmg.b [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Farmacia; Roux, Celine; Durand, Philippe; Iorga, Bogdan; Badet-Denisot, Marie A.; Badet, Bernard [Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette (France). Inst. de Chimie des Substances Naturelles

2010-07-01

The aminosugars are very important structural components of bacterial and fungi cell walls. Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from D-fructose-6-phosphate, is therefore an interesting target in the fight against microorganisms. In this work is described the synthesis of aromatic analogs of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) and its epimer 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP), two important inhibitors of GlmS. The aromatic analogs displayed modest inhibitory activity against GlmS, with IC{sub 50} in the mmol L{sup -1} range. (author)

Excessive fructose intake causes 1,25-(OH)2D3-dependent inhibition of intestinal and renal calcium transport in growing rats

Science.gov (United States)

Douard, Veronique; Sabbagh, Yves; Lee, Jacklyn; Patel, Chirag; Kemp, Francis W.; Bogden, John D.; Lin, Sheldon

2013-01-01

We recently discovered that chronic high fructose intake by lactating rats prevented adaptive increases in rates of active intestinal Ca2+ transport and in levels of 1,25-(OH)2D3, the active form of vitamin D. Since sufficient Ca2+ absorption is essential for skeletal growth, our discovery may explain findings that excessive consumption of sweeteners compromises bone integrity in children. We tested the hypothesis that 1,25-(OH)2D3 mediates the inhibitory effect of excessive fructose intake on active Ca2+ transport. First, compared with those fed glucose or starch, growing rats fed fructose for 4 wk had a marked reduction in intestinal Ca2+ transport rate as well as in expression of intestinal and renal Ca2+ transporters that was tightly associated with decreases in circulating levels of 1,25-(OH)2D3, bone length, and total bone ash weight but not with serum parathyroid hormone (PTH). Dietary fructose increased the expression of 24-hydroxylase (CYP24A1) and decreased that of 1α-hydroxylase (CYP27B1), suggesting that fructose might enhance the renal catabolism and impair the synthesis, respectively, of 1,25-(OH)2D3. Serum FGF23, which is secreted by osteocytes and inhibits CYP27B1 expression, was upregulated, suggesting a potential role of bone in mediating the fructose effects on 1,25-(OH)2D3 synthesis. Second, 1,25-(OH)2D3 treatment rescued the fructose effect and normalized intestinal and renal Ca2+ transporter expression. The mechanism underlying the deleterious effect of excessive fructose intake on intestinal and renal Ca2+ transporters is a reduction in serum levels of 1,25-(OH)2D3. This finding is significant because of the large amounts of fructose now consumed by Americans increasingly vulnerable to Ca2+ and vitamin D deficiency. PMID:23571713

Radiosynthesis, rodent biodistribution, and metabolism of 1-deoxy-1-[18F]fluoro-d-fructose

International Nuclear Information System (INIS)

Haradahira, Terushi; Tanaka, Akihiro; Maeda, Minoru; Kanazawa, Yoko; Ichiya, Yu-Ichi; Masuda, Kouji

1995-01-01

Fluorine-18 labeled analog of d-fructose, 1-deoxy-1-[ 18 F]fluoro-d-fructose (1-[ 18 F]FDFrc), was synthesized by nucleophilic substitution of [ 18 F]fluoride ion and the effect of the fluorine substitution on its in vivo metabolism was investigated. The tissue distributions of 1-[ 18 F]FDFrc in rats and tumor bearing mice showed initial high uptake and subsequent rapid washout of the radioactivity in the principal sites of d-fructose metabolism (kidneys, liver and small intestine). The uptakes in the brain and tumor (fibrosarcoma) were the lowest and moderate, respectively, but tended to increase with time. The in vivo metabolic studies of 1-[ 18 F]FDFrc and nonradiactive 1-FDFrc in mouse brain and tumor showed that the fluorinated analog remained unmetabolized in these tissues, indicating that the substitution of fluorine at the C-1 position produces a nonmetabolizable analog of d-fructose. Thus, 1-[ 18 F]FDFrc had no features of a metabolic trapping tracer without showing any appreciable organ or tumor specific localization

1,5-Anhydro-D-Fructose as Starting Material for the Synthesis of Biological Active Compounds

DEFF Research Database (Denmark)

Lundt, Inge; Andreassen, Mikkel; Maier, Peter

1,5-Anhydro-D-fructose (AF) is a valuable chiral building block[1,2] which is available by degradation from starch by a-1,4-glucan lyase[2] and which now also can be produced by efficient synthetic proedures.[3] AF has three different functional hydroxyl groups, a pro-chiral center and a permanent...... Ascopyrone P. [1] S. M. Andersen, I. Lundt, J. Marcussen and S.Yu, Carbohydr. Res. 337, 873 (2002). [2] EU-program NEPSA (NEw Products from Starch derived Anhydro-D-fructose) under the Cell Factory of 5th Framework Program, QLK3-2001-02400 [3] I. Lundt, M. Andreassen and A. Stütz, PA 2004 01060 [4] O. Deppe...

The Glycolytic Metabolite, Fructose-1,6-bisphosphate, Blocks Epileptiform Bursts by Attenuating Voltage-Activated Calcium Currents in Hippocampal Slices

Directory of Open Access Journals (Sweden)

Li-Rong Shao

2018-06-01

Full Text Available Manipulation of metabolic pathways (e.g., ketogenic diet (KD, glycolytic inhibition alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a “branching” metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP. Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5–5 mM blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min, suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531, nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA. In contrast, F1,6BP caused moderate reduction (~50% in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 μM, suggesting that these bursts are calcium dependent. Altogether, these data

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1.

Science.gov (United States)

Zhou, Yixing; Wu, Shilan; Wang, Huanchen; Hayakawa, Yoichi; Bird, Gary S; Shears, Stephen B

2012-12-01

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

Science.gov (United States)

Iversen, L F; Brzozowski, M; Hastrup, S; Hubbard, R; Kastrup, J S; Larsen, I K; Naerum, L; Nørskov-Lauridsen, L; Rasmussen, P B; Thim, L; Wiberg, F C; Lundgren, K

1997-05-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

[Fructose and fructose intolerance].

Science.gov (United States)

Buzás, György Miklós

2016-10-01

Although fructose was discovered in 1794, it was realised in recent decades only that its malabsorption can lead to intestinal symptoms while its excessive consumption induces metabolic disturbances. Fructose is a monosaccharide found naturally in most fruits and vegetables. Dietary intake of fructose has gradually increased in the past decades, especially because of the consumption of high fructose corn syrup. With its 16.4 kg/year consumption, Hungary ranks secondly after the United States. Fructose is absorbed in the small intestine by facilitated transport mediated by glucose transporter proteins-2 and -5, and arrives in the liver cells. Here it is transformed enzymatically into fructose-1-phosphate and then, fructose-1,5-diphosphate, which splits further into glyceraldehyde and dihydroxyacetone-phosphate, entering the process of glycolysis, triglyceride and uric acid production. The prevalence of fructose intolerance varies strongly, depending on the method used. The leading symptoms of fructose intolerance are similar, but less severe than those of lactose intolerance. Multiple secondary symptoms can also occur. A symptom-based diagnosis of fructose intolerance is possible, but the gold standard is the H 2 breath test, though this is less accurate than in lactose testing. Measuring fructosaemia is costly, cumbersome and not widely used. Fructose intolerance increases intestinal motility and sensitivity, promotes biofilm formation and contributes to the development of gastrooesophageal reflux. Long-term use of fructose fosters the development of dental caries and non-alcoholic steatohepatitis. Its role in carcinogenesis is presently investigated. The cornerstone of dietary management for fructose intolerance is the individual reduction of fructose intake and the FODMAP diet, led by a trained dietetician. The newly introduced xylose-isomerase is efficient in reducing the symptoms of fructose intolerance. Orv. Hetil., 2016, 157(43), 1708-1716.

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

Directory of Open Access Journals (Sweden)

Roberto P Stock

Full Text Available The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1 ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2 the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3 in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

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Kumar, Arvind; Rai, Lal Chand

2015-01-01

Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes. Copyright © 2014 Elsevier GmbH. All rights reserved.

D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens

International Nuclear Information System (INIS)

Lessmann, D.; Schimz, K.L.; Kurz, G.

1975-01-01

The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300μmol NADH formed per min per mg protein, was shown to be homogeneous. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220,000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265,000. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal acivity at pH 8.9. The Entner-Doudoroff enzyme showed specificity for NAD + as well as for NADP + and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of β-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase. (orig.) [de

Structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi

International Nuclear Information System (INIS)

Gardberg, Anna; Sankaran, Banumathi; Davies, Doug; Bhandari, Janhavi; Staker, Bart; Stewart, Lance

2011-01-01

The eukaryotic parasite E. cuniculi expresses a fructose bisphosphate aldolase that crystallizes readily in the presence of the partial substrate analog phosphate. This aldolase–phosphate structure and that of the sugar-bound Schiff base are reported. E. cuniculi aldolase displays a dimeric structure rather than the expected tetrameric quaternary structure. Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO 4 , 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml −1 and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 121.46, b = 135.82, c = 61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C222 1 and the unit-cell parameters were a = 121.96, b = 137.61, c = 62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental

Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis.

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Ganapathy, Uday; Marrero, Joeli; Calhoun, Susannah; Eoh, Hyungjin; de Carvalho, Luiz Pedro Sorio; Rhee, Kyu; Ehrt, Sabine

2015-08-10

The human pathogen Mycobacterium tuberculosis (Mtb) likely utilizes host fatty acids as a carbon source during infection. Gluconeogenesis is essential for the conversion of fatty acids into biomass. A rate-limiting step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate by a fructose bisphosphatase (FBPase). The Mtb genome contains only one annotated FBPase gene, glpX. Here we show that, unexpectedly, an Mtb mutant lacking GLPX grows on gluconeogenic carbon sources and has detectable FBPase activity. We demonstrate that the Mtb genome encodes an alternative FBPase (GPM2, Rv3214) that can maintain gluconeogenesis in the absence of GLPX. Consequently, deletion of both GLPX and GPM2 is required for disruption of gluconeogenesis and attenuation of Mtb in a mouse model of infection. Our work affirms a role for gluconeogenesis in Mtb virulence and reveals previously unidentified metabolic redundancy at the FBPase-catalysed reaction step of the pathway.

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose.

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Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

2007-11-23

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 A, respectively. A subunit of P. cichoriid-TE adopts a (beta/alpha)(8) barrel structure, and a metal ion (Mn(2+)) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the beta-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn(2+), and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.

1,5-Anhydro-D-fructose as Chiral Building Block: A Novel Approach to 1-Deoxymannojirimycin

DEFF Research Database (Denmark)

Maier, Peter; Andersen, Søren Møller; Lundt, Inge

2006-01-01

A novel six-step synthesis of 1-deoxymannojirimycin from 1,5-anhydro-D-fructose in 35% overall yield is reported. The key steps are nucleophilic piperidine ring formation and subsequent Lewis acid induced pyran ether cleavage.......A novel six-step synthesis of 1-deoxymannojirimycin from 1,5-anhydro-D-fructose in 35% overall yield is reported. The key steps are nucleophilic piperidine ring formation and subsequent Lewis acid induced pyran ether cleavage....

Outer membrane-localised fructose 1,6-bisphosphate aldolase of ...

African Journals Online (AJOL)

user

Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom. Accepted 6 June, 2012. Fructose-1,6-bisphosphate aldolase (FBA) is a classical cytoplasmic glycolytic enzyme which, despite lacking a predicted signal peptide, has been demonstrated to be expressed and transported to ...

Enzymatic production of microthecin by aldos-2-ulose dehydratase from 1,5-anhydro-D-fructose and stability studies of microthecin

DEFF Research Database (Denmark)

Yu, Shukun; Andreassen, Mikkel; Lundt, Inge

2008-01-01

Microthecin (2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one), which has anti-microbial activity, is one of the end products of an alternative glycogen and starch degrading pathway, the anhydrofructose pathway. It is formed from 1,5- anhydro-D-fructose (AF), a product of a-1,4-glucan lyase (EC 4...

Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose.

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Zhang, Longtao; Mu, Wanmeng; Jiang, Bo; Zhang, Tao

2009-06-01

A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased for other substrates in the order: D-tagatose, D-psicose, D-ribulose, D-xylulose and D-sorbose. Its activity was maximal at pH 9 and 40 degrees C while being enhanced by Mn(2+). At pH 9 and 40 degrees C, 118 g D-psicose l(-1) was produced from 700 g D-fructose l(-1) after 3 h.

Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum

Directory of Open Access Journals (Sweden)

Jeffrey Thomas Cole

2012-04-01

Full Text Available Brain cells expend large amounts of energy sequestering calcium (Ca2+, while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P, a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA. Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1-10 mM. The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity

Effects of oral-D-tagatose, A stereoisomer of D-fructose, on liver metabolism in man as examined by 31P-magnetic resonance spectroscopy

DEFF Research Database (Denmark)

Buemann, B.; Toubro, S.; Gesmar, H.

2000-01-01

in inorganic phosphate (Pi) and adenosine triphosphate (ATP) levels in the liver compared with D-fructose. Similar to what is seen in patients with hereditary fructose intolerance, this may increase purine nucleotide degradation and thereby increase uric acid production. The effect of 30 g D-tagatose or D...... equivalent to about 1 mmol/L was found in the spectrum within 30 minutes after D-tagatose was administered in all subjects. Concomitantly, ATP was reduced by about 12% (P effects had vanished after 150 minutes. Serum uric acid concentration was increased by 17% 50 minutes after D-tagatose (P...... effect of D-tagatose. No changes in 31PMRS spectra or serum uric acid...

Vanadate influence on metabolism of sugar phosphates in fungus Phycomyces blakesleeanus.

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Milan Žižić

Full Text Available The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+ on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC, and polyphosphates, UDPG and ATP (31P NMR was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+, indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

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Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

2014-06-01

The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

3D-QSAR studies and molecular docking on [5-(4-amino-1 H-benzoimidazol-2-yl)-furan-2-yl]-phosphonic acid derivatives as fructose-1,6-biphophatase inhibitors

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Lan, Ping; Xie, Mei-Qi; Yao, Yue-Mei; Chen, Wan-Na; Chen, Wei-Min

2010-12-01

Fructose-1,6-biphophatase has been regarded as a novel therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). 3D-QSAR and docking studies were performed on a series of [5-(4-amino-1 H-benzoimidazol-2-yl)-furan-2-yl]-phosphonic acid derivatives as fructose-1,6-biphophatase inhibitors. The CoMFA and CoMSIA models using thirty-seven molecules in the training set gave r cv 2 values of 0.614 and 0.598, r 2 values of 0.950 and 0.928, respectively. The external validation indicated that our CoMFA and CoMSIA models possessed high predictive powers with r 0 2 values of 0.994 and 0.994, r m 2 values of 0.751 and 0.690, respectively. Molecular docking studies revealed that a phosphonic group was essential for binding to the receptor, and some key features were also identified. A set of forty new analogues were designed by utilizing the results revealed in the present study, and were predicted with significantly improved potencies in the developed models. The findings can be quite useful to aid the designing of new fructose-1,6-biphophatase inhibitors with improved biological response.

2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate.

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Takanori Nihira

Full Text Available The glycoside hydrolase family (GH 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816 from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi-dependent hydrolysis of kojibiose at the rate of 0.43 s(-1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-D-glucose 1-phosphate (βGlc1P as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a k cat of 2.8 s(-1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 (18O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-D-glucosylglycerol (GG at the rate of 180 s(-1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s(-1. We propose 2-O-α-D-glucopyranosylglycerol: phosphate β-D-glucosyltransferase as the systematic name and 2-O-α-D-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

1,5-Anhydro-D-fructose: biocatalytic and chemical synthetic methods for the preparation, transformation and derivatization

DEFF Research Database (Denmark)

Lundt, Inge; Yu, Shukun

2010-01-01

1,5-Anhydro-D-fructose (1,5AnFru) is a monoketosaccharide that can be prepared enzymatically from starch by a-1,4-glucan lyase or chemically from D-glucose or D-fructose in a few steps with high yields. The formed 1,5AnFru can be derivatized both enzymatically and chemically to interesting new...

Radiation-induced degradation of D-fructose in aerated condition

International Nuclear Information System (INIS)

Kito, Yukio; Kawakishi, Shunro; Namiki, Mitsuo

1981-01-01

Gamma-radiolysis of fructose in aqueous solution under aerated conditions formed various oxidized products, such as dicarbonyl hexoses, lower molecular aldoses and aldonic acids. Among these radiolytic products, D-arabinohexosulose (1, G = 2.2) and D-threo-2,5-hexodiulose (2, G = 1.5) were identified as major hexose derivatives, and D-threo-2,3-hexodiulose (3) and D-lyxo-hexos-5-ulose (4) as minor products. The radiolytic processes were found to be derived through fructose radicals, similarly to anaerobic radiolysis of fructose. The mechanism of radiolysis was proposed to be initiated by hydrogen abstraction with hydroxyl radical, followed by formation and degradation of fructose hydroperoxy radicals. (author)

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

International Nuclear Information System (INIS)

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Daniele; Fagret, Daniel; Ghezzi, Catherine; Halimi, Serge; Demongeot, Jacques

2007-01-01

Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state that has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats using 125 I-6-deoxy-6-iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic-normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood was assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p < 0.01; muscle, p < 0.05; heart, p < 0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p < 0.001; muscle, p < 0.001; heart, p < 0.01) whereas no significant changes were observed in fructose-fed rats. This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. (orig.)

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

Science.gov (United States)

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Danièle; Halimi, Serge; Demongeot, Jacques; Fagret, Daniel; Ghezzi, Catherine

2007-01-01

Purpose Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state and it has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats with 125I-6-Deoxy-6-Iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Methods Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood were assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Results Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady-state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p<0.01; muscle, p<0.05; heart, p<0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p<0.001; muscle, p<0.001; heart, p<0.01) and whereas no significant changes were observed in fructose-fed rats. Conclusion This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. PMID:17171359

1,5-Anhydro-D-Fructose – Efficient Synthesis and Chemical Uses

DEFF Research Database (Denmark)

Lundt, Inge; Dekany, Gyula; Stütz, Arnold E.

as well as for derivatives and analogues thereof. The potential of AF will be highlighted as will be the use of AF as a chiral building block for the preparation of other interesting compounds with biological activities such as, for example, Deoxymannojirimycin (DMJ). [1] S.M. Andersen, I. Lundt, J......1,5-Anhydro-D-fructose (AF) is a valuable chiral building block for organic synthesis.[1] However, the antioxidant and antimicrobial properties of AF are equally important.[1] Due to these interesting properties AF is heavily patented for the use in pharmaceuticals, foods and cosmetics. However...

Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

International Nuclear Information System (INIS)

Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

2010-01-01

Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

Science.gov (United States)

Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

1992-01-01

The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

International Nuclear Information System (INIS)

Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B.

1989-01-01

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue

Assessment of insulin resistance in fructose-fed rats with {sup 125}I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

Energy Technology Data Exchange (ETDEWEB)

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Daniele; Fagret, Daniel; Ghezzi, Catherine [INSERM, E340, 38000 Grenoble, (France); Univ Grenoble, 38000 Grenoble, (France); Halimi, Serge [CHRU Grenoble, Hopital Michallon, Service de Diabetologie, 38000 Grenoble, (France); Demongeot, Jacques [Univ Grenoble, 38000 Grenoble, (France); CNRS, UMR 5525, 38000 Grenoble, (France)

2007-05-15

Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state that has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats using {sup 125}I-6-deoxy-6-iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic-normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood was assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p < 0.01; muscle, p < 0.05; heart, p < 0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p < 0.001; muscle, p < 0.001; heart, p < 0.01) whereas no significant changes were observed in fructose-fed rats. This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. (orig.)

Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli

International Nuclear Information System (INIS)

Iino, Daisuke; Takakura, Yasuaki; Kuroiwa, Mika; Kawakami, Ryouta; Sasaki, Yasuyuki; Hoshino, Takayuki; Ohsawa, Kanju; Nakamura, Akira; Yajima, Shunsuke

2007-01-01

The crystallization and preliminary X-ray studies of the aminoglycoside antibiotic-modifying enzyme hygromycin B phosphotransferase from E. coli are reported. Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 2.7.1.119) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3 2 21, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein

Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Boyko, Konstantin M., E-mail: kmb@inbi.ras.ru [Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow (Russian Federation); National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation); Gorbacheva, Marina A.; Korzhenevskiy, Dmitry A. [National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation); Alekseeva, Maria G.; Mavletova, Dilara A.; Zakharevich, Natalia V.; Elizarov, Sergey M.; Rudakova, Natalia N.; Danilenko, Valery N. [Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina str. 3, Moscow, 119333 (Russian Federation); Popov, Vladimir O. [Bach Institute of Biochemistry, Federal Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky Prospekt. 33, Bld. 2, 119071, Moscow (Russian Federation); National Research Center “Kurchatov Institute”, Kurchatov Complex of NBICS-technologies, Akad. Kurchatova sqr., 1, Moscow, 123182 (Russian Federation)

2016-09-02

Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3′-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. - Highlights: • 3D structure of the novel aminoglycoside phosphotransferase AphVIII was obtained. • AphVIII activation loop is clearly identified in the electron density. • AphVIII has some unique structural features in its substrate C-ring binding pocket.

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate

DEFF Research Database (Denmark)

Stock, Roberto; Brewer, Jonathan R.; Wagner, Kerstin

2012-01-01

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model...... membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy...... and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing...

Radiopharmacological evaluation of 6-deoxy-6-[{sup 18}F]fluoro-D-fructose as a radiotracer for PET imaging of GLUT5 in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Wuest, Melinda, E-mail: mwuest@ualberta.c [Department of Oncology, University of Alberta - Cross Cancer Institute, Edmonton, AB-T6G 1Z2 (Canada); Trayner, Brendan J. [Department of Physiology, University of Alberta, Edmonton, AB-T6G 1Z2 (Canada); Grant, Tina N. [Department of Physiology, University of Alberta, Edmonton, AB-T6G 1Z2 (Canada); Department of Chemistry, University of Alberta, Edmonton, AB-T6G 1Z2 (Canada); Jans, Hans-Soenke; Mercer, John R.; Murray, David [Department of Oncology, University of Alberta - Cross Cancer Institute, Edmonton, AB-T6G 1Z2 (Canada); West, Frederick G. [Department of Chemistry, University of Alberta, Edmonton, AB-T6G 1Z2 (Canada); McEwan, Alexander J.B.; Wuest, Frank [Department of Oncology, University of Alberta - Cross Cancer Institute, Edmonton, AB-T6G 1Z2 (Canada); Cheeseman, Chris I. [Department of Physiology, University of Alberta, Edmonton, AB-T6G 1Z2 (Canada)

2011-05-15

Introduction: Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[{sup 18}F]fluoro-D-glucose ([{sup 18}F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[{sup 18}F]fluoro-D-fructose (6-[{sup 18}F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers. Methods: Uptake of 6-[{sup 18}F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [{sup 18}F]FDG. Biodistribution of 6-[{sup 18}F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [{sup 18}F]FDG. 6-[{sup 18}F]FDF metabolism was investigated in mouse blood and urine. Results: 6-[{sup 18}F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30{+-}4% (n=9) and 12{+-}1% (n=7) ID/mg protein 6-[{sup 18}F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-D-fructose had a 10-fold higher potency than fructose to inhibit 6-[{sup 18}F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65{+-}0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75{+-}0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0

GATA4-mediated cardiac hypertrophy induced by D-myo-inositol 1,4,5-tris-phosphate

International Nuclear Information System (INIS)

Zhu Zhiming; Zhu Shanjun; Liu Daoyan; Yu Zengping; Yang Yongjian; Giet, Markus van der; Tepel, Martin

2005-01-01

We evaluated the effects of D-myo-inositol 1,4,5-tris-phosphate on cardiac hypertrophy. D-myo-inositol 1,4,5-tris-phosphate augmented cardiac hypertrophy as evidenced by its effects on DNA synthesis, protein synthesis, and expression of immediate-early genes c-myc and c-fos, β-myosin heavy chain, and α-actin. The administration of D-myo-inositol 1,4,5-tris-phosphate increased the expression of nuclear factor of activated T-cells and cardiac-restricted zinc finger transcription factor (GATA4). Real-time quantitative RT-PCR showed that D-myo-inositol 1,4,5-tris-phosphate-induced GATA4 mRNA was significantly enhanced even in the presence of the calcineurin inhibitor, cyclosporine A. The effect of D-myo-inositol 1,4,5-tris-phosphate was blocked after inhibition of inositol-trisphosphate receptors but not after inhibition of c-Raf/mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (ERK) or p38 mitogen-activated protein kinase pathways. The study shows that D-myo-inositol 1,4,5-tris-phosphate-induced cardiac hypertrophy is mediated by GATA4 but independent from the calcineurin pathway

Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

Science.gov (United States)

Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

2006-01-01

The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%. PMID:16461638

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO2 levels.

Science.gov (United States)

Tamoi, Masahiro; Hiramatsu, Yoshie; Nedachi, Shigeki; Otori, Kumi; Tanabe, Noriaki; Maruta, Takanori; Shigeoka, Shigeru

2011-05-01

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO(2) levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO(2) levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO(2) levels.

Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

International Nuclear Information System (INIS)

Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose

2008-01-01

The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(d-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from d-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethyl phosphoryl chloride. The resulting 5-[d-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase. (author)

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes.

Science.gov (United States)

Honda, Kohsuke; Maya, Shohei; Omasa, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Ohtake, Hisao

2010-08-02

Six thermophilic enzymes from Thermus thermophilus were used to construct an 'artificial bio-synthetic pathway' for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%. Copyright 2010 Elsevier B.V. All rights reserved.

Synthesis of naturally occurring iminosugars from D-fructose by the use of a zinc-mediated fragmentation reaction

DEFF Research Database (Denmark)

Lauritsen, Anne; Madsen, Robert

2006-01-01

A short synthesis of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and a formal synthesis of australine are described. In both cases, D-fructose is employed as the starting material and converted into a protected methyl 6-deoxy-6-iodo-furanoside. Zinc-mediated fragmentation produces an unsaturated...... ketone which serves as a key building block for both syntheses. Ozonolysis, reductive amination with benzylamine and deprotection affordfs 1,4-dideoxy-1,4-imino-D-arabinitol in only 7 steps and 11% overall yield from D-fructose. Alternatively, reductive amination with homoallylamine, ring......-closing metathesis and protecting group manipulations give rise to an intermediate which can be converted into australine in 3 steps. The intermediate is prepared by two different strategies both of which use a total of 9 steps. The first strategy utilizes benzyl ethers for protection of fructose while the second...

Fructose in Breast Milk Is Positively Associated with Infant Body Composition at 6 Months of Age.

Science.gov (United States)

Goran, Michael I; Martin, Ashley A; Alderete, Tanya L; Fujiwara, Hideji; Fields, David A

2017-02-16

Dietary sugars have been shown to promote excess adiposity among children and adults; however, no study has examined fructose in human milk and its effects on body composition during infancy. Twenty-five mother-infant dyads attended clinical visits to the Oklahoma Health Sciences Center at 1 and 6 months of infant age. Infants were exclusively breastfed for 6 months and sugars in breast milk (i.e., fructose, glucose, lactose) were measured by Liquid chromatography-mass spectrometry (LC-MS/MS) and glucose oxidase. Infant body composition was assessed using dual-energy X-ray absorptiometry at 1 and 6 months. Multiple linear regression was used to examine associations between breast milk sugars and infant body composition at 6 months of age. Fructose, glucose, and lactose were present in breast milk and stable across visits (means = 6.7 μg/mL, 255.2 μg/mL, and 7.6 g/dL, respectively). Despite its very low concentration, fructose was the only sugar significantly associated with infant body composition. A 1-μg/mL higher breast milk fructose was associated with a 257 g higher body weight ( p = 0.02), 170 g higher lean mass ( p = 0.01), 131 g higher fat mass ( p = 0.05), and 5 g higher bone mineral content ( p = 0.03). In conclusion, fructose is detectable in human breast milk and is positively associated with all components of body composition at 6 months of age.

The Effects of Fenarimol and Methyl Parathion on Glucose 6-Phosphate Dehydrogenase Enzyme Activity in Rats

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Ferda ARI

2017-10-01

Full Text Available Fenarimol and methyl parathion are pesticides that have been used in agriculture for several years. These pesticides have significant effects on environmental and human health. Therefore, we investigated the effects of methyl parathion and fenarimol on glucose 6-phosphate dehydrogenase (EC 1.1.1.49 enzyme activity in rats. The glucose 6- phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway and it is important in detoxifying reactions by NADPH generated. In this study, wistar albino rats administrated with methyl parathion (7 mg kg–1 and fenarimol (200 mg kg−1 by intraperitoneally for different periods (2, 4, 8, 16, 32, 64, and 72 h. The glucose 6-phosphate dehydrogenase enzyme activity was assayed in liver, kidney, brain, and small intestine in male and female rats. The exposure of fenarimol and methyl parathion caused increase of glucose 6-phosphate dehydrogenase enzyme activity in rat tissues, especially at last periods. We suggest that this increment of enzyme activity may be the reason of toxic effects of fenarimol and methyl parathion.

Effect of ionic liquids, 1-butyl-3-methyl imidazolium bromide and 1-hexyl-3-methyl imidazolium bromide on the vapour – Liquid equilibria of the aqueous D-fructose solutions at 298.15 K and atmospheric pressure using isopiestic method

International Nuclear Information System (INIS)

Zafarani-Moattar, Mohammed Taghi; Shekaari, Hemayat; Mazaher Haji Agha, Elnaz

2017-01-01

Highlights: • VLE data for aqueous fructose + [BMIm]Br or [HMIm]Br systems were measured. • Performances of different local composition models were tested in fitting VLE data. • Molal activity coefficients were calculated. • The results were discussed on basis of water, IL and sugar interactions. - Abstract: In this study, water activity measurements have been carried out by the isopiestic method for the systems (D-fructose + 1-butyl-3-methyl imidazolium bromide + H 2 O) and (D-fructose + 1-hexyl-3-methyl imidazolium bromide + H 2 O) at 298.15 K and atmospheric pressure. Vapour pressures and osmotic coefficients of the solutions have been determined from the experimental measured water activity results. The experimental water activity values were satisfactorily correlated with segment-based local composition models of the Wilson, NRTL, modified NRTL, NRF-NRTL and UNIQUAC. Then, using the parameters obtained from these models, the unsymmetrical molal activity coefficients of the D-fructose and ionic liquids in the binary and D-fructose in ternary aqueous solutions have been calculated. Furthermore, the activity coefficients of D-fructose in binary and ternary solutions were used to calculate the Gibbs energy of transfer for D-fructose from water to aqueous ionic liquid solutions. An application of McMillan-Mayer theory of solutions through virial expansion of transfer Gibbs energy was made to get pair and triplet interaction parameters and salting constant values. From the sign and magnitude of these parameters and salting constants and also from the magnitude of activity coefficients some information about solute-solute and solute-solvent interactions are obtained.

Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

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Jin-Suk Han

2005-01-01

Full Text Available The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261 is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

Antioxidant activity of the melanoidin fractions formed from DGlucose and D-Fructose with L-Asparagine in the Maillard reaction

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A.P Echavarría

2013-03-01

Full Text Available Melanoidins formed at the last stage of the Maillard reaction have been shown to possess certain functional properties, such as antioxidant activity. In order to gain more insight into these functional properties, soluble model systems melanoidins from L‑Asparagine with D‑glucose or D‑fructose fractionating by ultrafiltration were analyzed. The fractionating/concentration sequence of the melanoidin fraction (1-300 kDa enabled five fractions to be produced.Additionally, the absorption of melanoidins was measured at different wavelengths (280, 325, 405 and browning at 420 nm. The fractionation effect of melanoidin systems on the color intensity, UV-absorbance scan wavelengths (nm, CIE, L*, a*, b* parameters and antioxidant activity were measured. For this purpose, antioxidant activity was evaluated through the free radical scavenging activity, including 1,1-diphenyl-2-picryl-hydrazil (DPPH and 2,20-azinobis (3-ethylbenothiazoline-6-sulfonic acid, diammonium salt (ABTS. The results showed that the absorption of the melanoidins formed from Glucose/L-Asn was higher than for those derived from Fructose/L-Asn. On the other hand, their antioxidant power was lower than that for melanoidins formed from Fructose/L-Asn systems.

The Effect of D-Tagatose on Fructose Absorption in a Rat Model.

Science.gov (United States)

Williams, Jarrod; Spitnale, Michael; Lodder, Robert

D-tagatose is in development as a medication for the treatment of type 2 diabetes. The effect of oral D-tagatose on the absorption of D-fructose was assessed when co-administered in this study. In the pilot study, male Sprague-Dawley rats were fed C14 labeled fructose and glucose concomitantly to establish dose levels for the treatment group of rats fed C14 labeled fructose together with D-tagatose. Rats were administered 0, 600, 2000, 6000, or 12000 mg/kg of D-tagatose along with 2000 mg/kg of fructose. Blood samples were taken over 60 minutes and were assessed using scintillation counting. 600, 2000, and 6000 mg/kg of D-tagatose decreased fructose absorption by 1%, 26%, and 30% respectively (12000 mg/kg group was stopped short of completion due to intolerance) as measured by AUC of scintillation counts. The 600 and 2000 mg/kg of D-tagatose groups showed no difference in plasma glucose concentrations compared to placebo while a rise in glucose was seen in the 6000 mg/kg of D-tagatose groups. The results indicate that D-tagatose may be useful in reducing fructose absorption, which could lead to a beneficial outcome.

D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate, a mimic of D-myo-inositol 1,3,4,5-tetrakisphosphate: biological activity and pH-dependent conformational properties

International Nuclear Information System (INIS)

Horne, Graeme; Maechling, Clarisse; Fleig, Andrea; Hirata, Masato; Penner, Reinhold; Spiess, Bernard; Potter, Barry V.L.

2004-01-01

D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate [D-6-deoxy-Ins(1,3,4,5)P 4 ] 3 is a novel deoxygenated analogue of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P 4 ] 2, a central and enigmatic molecule in the polyphosphoinositide pathway of cellular signalling. D-6-Deoxy-Ins(1,3,4,5)P 4 is a moderate inhibitor of Ins(1,4,5)P 3 5-phosphatase [1.8 μM] compared to Ins(1,3,4,5)P 4 [0.15 μM] and similar to that of L-Ins(1,3,4,5)P 4 [1.8 μM]. In displacement of [ 3 H] Ins(1,4,5)P 3 from the rat cerebellar Ins(1,4,5)P 3 receptor, while slightly weaker [IC 50 =800 nM] than that of D-Ins(1,3,4,5)P 4 [IC 50 =220 nM], 3 is less markedly different and again similar to that of L-Ins(1,3,4,5)P 4 [IC 50 =660 nM]. 3 is an activator of I CRAC when inward currents are measured in RBL-2H3-M1 cells using patch-clamp electrophysiological techniques with a facilitation curve different to that of Ins(1,3,4,5)P 4 . Physicochemical properties were studied by potentiometric 31 P and 1 H NMR titrations and were similar to those of Ins(1,3,4,5)P 4 apart from the observation of a biphasic titration curve for the P1 phosphate group. A novel vicinal phosphate charge-induced conformational change of the inositol ring above pH 10 was observed for D-6-deoxy-Ins(1,3,4,5)P 4 that would normally be hindered because of the central stabilising role played by the 6-OH group in Ins(1,3,4,5)P 4 . We conclude that the 6-OH group in Ins(1,3,4,5)P 4 is crucial for its physicochemical behaviour and biological properties of this key inositol phosphate

Regulation by S-nitrosylation of the Calvin-Benson cycle fructose-1,6-bisphosphatase in Pisum sativum

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Antonio Jesús Serrato

2018-04-01

Full Text Available Redox regulation is of great importance in chloroplasts. Many chloroplast enzymes, such as those belonging to the Calvin-Benson cycle (CBC, have conserved regulatory cysteines which form inhibitory disulphide bridges when physiological conditions become unfavourable. Amongst these enzymes, cFBP1, the CBC fructose-1,6-bisphosphatase (FBPase isoform, is well known to be redox activated by thioredoxin f through the reduction of a disulphide bridge involving Cys153 and Cys173. Moreover, data obtained during recent years point to S-nitrosylation as another redox post-translational modification putatively regulating an increasing number of plant enzymes, including cFBP1. In this study we have shown that the Pisum sativum cFBP1 can be efficiently S-nitrosylated by GSNO and SNAP, triggering the formation of the regulatory disulphide. Using in vivo experiments with P. sativum we have established that cFBP1 S-nitrosylation only occurs during the light period and we have elucidated by activity assays with Cys-to-Ser mutants that this enzyme may be inactivated through the S-nitrosylation of Cys153. Finally, in the light of the new data, we have proposed an extended redox-regulation model by integrating the S-nitrosylation and the TRX f-mediated regulation of cFBP1. Keywords: S-nitrosylation, GSNO, Redox regulation, Fructose-1,6-bisphosphatase, Pisum sativum, Calvin-Benson cycle

Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup.

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Singh, Ram Sarup; Dhaliwal, Rajesh; Puri, Munish

2007-05-01

An extracellular exoinulinase (2,1-beta-D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable (100%) for 3 h at the optimum temperature of 50 degrees C. Mn2+ and Ca2+ produced a 2.4-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6 mg/ml and 41.3 mg/ml, respectively.

The Effect of D-Tagatose on Fructose Absorption in a Rat Model

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Williams, Jarrod; Spitnale, Michael; Lodder, Robert

2014-01-01

D-tagatose is in development as a medication for the treatment of type 2 diabetes. The effect of oral D-tagatose on the absorption of D-fructose was assessed when co-administered in this study. In the pilot study, male Sprague-Dawley rats were fed C14 labeled fructose and glucose concomitantly to establish dose levels for the treatment group of rats fed C14 labeled fructose together with D-tagatose. Rats were administered 0, 600, 2000, 6000, or 12000 mg/kg of D-tagatose along with 2000 mg/kg of fructose. Blood samples were taken over 60 minutes and were assessed using scintillation counting. 600, 2000, and 6000 mg/kg of D-tagatose decreased fructose absorption by 1%, 26%, and 30% respectively (12000 mg/kg group was stopped short of completion due to intolerance) as measured by AUC of scintillation counts. The 600 and 2000 mg/kg of D-tagatose groups showed no difference in plasma glucose concentrations compared to placebo while a rise in glucose was seen in the 6000 mg/kg of D-tagatose groups. The results indicate that D-tagatose may be useful in reducing fructose absorption, which could lead to a beneficial outcome. PMID:25621289

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

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Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-05

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. Copyright © 2016 Elsevier B.V. All rights reserved.

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

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Thompson, J.; Chassy, B.M.; Egan, W.

1985-01-01

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of [ 14 C]lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution 31 P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM

Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

Energy Technology Data Exchange (ETDEWEB)

Thompson, J.; Chassy, B.M.; Egan, W.

1985-04-01

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

Differential effects of acute and chronic fructose administration on pyruvate dehydrogenase activity and lipogenesis

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Wilson, L.

1988-01-01

These studies were undertaken to distinguish between the acute and chronic effects of fructose administration. In vivo, liver lipogenesis, as measured by 3 H 2 O incorporation, was greater in rats fed 60% fructose than in their glucose fed controls. Both fructose feeding, and fructose feeding plus intraperitoneal fructose injection increased the activities of 6-phosphogluconate dehydrogenase and malic enzyme. Liver PDH activity was increased by fructose feeding, and was increased even more by fructose feeding and injection of fructose, but this was not associated with any changes in hepatic ATP concentrations

1,5-Anhydro-D-fructose attenuates lipopolysaccharide-induced cytokine release via suppression of NF-κB p65 phosphorylation

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Meng Xiaojie; Kawahara, Ko-ichi; Nawa, Yuko; Miura, Naoki; Shrestha, Binita; Tancharoen, Salunya; Sameshima, Hisayo; Hashiguchi, Teruto; Maruyama, Ikuro

2009-01-01

Lipopolysaccharide (LPS) stimulates macrophages by activating NF-κB, which contributes to the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6. 1,5-anhydro-D-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits anti-oxidant activity and enhances insulin secretion. This study examined the effects of 1,5-AF on LPS-induced inflammatory reactions and elucidated its molecular mechanisms. Before LPS challenge, mice were pretreated with 1,5-AF (38.5 mg/kg). We found that 1,5-AF pretreatment attenuated cytokine release into the serum, including TNF-α, IL-6 and macrophage chemoattractant protein (MCP)-1. Furthermore, pretreatment with 1,5-AF (500 μg/ml) attenuated cytokine release, and 1,5-AF directly inhibited the nuclear translocalization of the NF-κB p65 subunit in LPS-stimulated murine macrophage-like RAW264.7 cells. This inhibition was responsible for decreased LPS-induced phosphorylation on Ser536 of the NF-κB p65 subunit, which is a posttranslational modification involved in the non-canonical pathway. Collectively, these findings indicate that the anti-inflammatory activity of 1,5-AF occurs via inactivation of NF-κB.

Dioscoreophyllum cumminsii (Stapf) Diels leaves halt high-fructose induced metabolic syndrome: Hyperglycemia, insulin resistance, inflammation and oxidative stress.

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Ajiboye, T O; Aliyu, H; Tanimu, M A; Muhammad, R M; Ibitoye, O B

2016-11-04

Dioscoreophyllum cumminsii is widely used in the management and treatment of diabetes and obesity in Nigeria. This study evaluates the effect of aqueous leaf extract of D. cumminsii on high-fructose diet-induced metabolic syndrome. Seventy male rats were randomized into seven groups. All rats were fed with high-fructose diet for 9 weeks except groups A and C rats, which received control diet. In addition to the diet treatment, groups A and B rats received distilled water for 3 weeks starting from the seventh week of the experimental period. Rats in groups C-F orally received 400, 100, 200 and 400mg/kg body weight of aqueous leaf extract of D. cumminsii respectively, while group G received 300mg/kg bodyweight of metformin for 3 weeks starting from the seventh week. There was significant (phigh-fructose diet-mediated increase in body weight, body mass index, abdominal circumference, blood glucose, insulin, leptin and insulin resistance by aqueous leaf extract of D. cumminsii. Conversely, high-fructose diet-mediated decrease in adiponectin was reversed by the extract. Increased levels of cholesterol, triglycerides, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, atherogenic index, cardiac index and coronary artery index were significantly lowered by the extract, while high-fructose diet mediated decrease in high-density lipoprotein cholesterol was increased by the extract. Tumour necrosis factor-α, interleukin-6 and interleukin-8 levels increased significantly in high-fructose diet-fed rats, which were significantly reversed by the extract. High-fructose mediated-decrease in superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and glutathione reduced were significantly reversed by aqueous leaf extract of D. cumminsii. Conversely, elevated levels of malondialdehyde, conjugated dienes, lipid hydroperoxides, protein carbonyl and fragmented DNA were significantly lowered by the

D-psicose, an epimer of D-fructose, favorably alters lipid metabolism in Sprague-Dawley rats.

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Nagata, Yasuo; Kanasaki, Akane; Tamaru, Shizuka; Tanaka, Kazunari

2015-04-01

D-Psicose, a C3 epimer of D-fructose, is known to lower body weight and adipose tissue weight and affect lipid metabolism. The precise mechanism remains unknown. It has been reported that D-psicose has a short half-life and is not metabolized in the body. To determine how D-psicose modifies lipid metabolism, rats were fed diets with or without 3% D-psicose for 4 weeks. Rats were decapitated without fasting every 6 h over a period of 24 h. Changes in serum and liver lipid levels, liver enzyme activity, and gene expression were quantified in experiment 1. Rats fed D-psicose had significantly lower serum insulin and leptin levels. Liver enzyme activities involved in lipogenesis were significantly lowered by the D-psicose diet, whereas gene expression of a transcriptional modulator of fatty acid oxidation was enhanced. In experiment 2, feeding the D-psicose diet gave significantly lower body weight (389 ± 3 vs 426 ± 6 g, p vs 25.7 ± 0.4 g/day, p energy expenditure in the light period and fat oxidation in the dark period compared to rats fed the control diet, whereas carbohydrate oxidation was lower. In summary, these results indicate that the D-psicose diet decreases lipogenesis, increases fatty acid oxidation, and enhances 24 h energy expenditure, leading to d-psicose's potential for weight management.

Initiation of proteolysis of yeast fructose-1,6-bisphosphatase by pH-control of adenylate cyclase

International Nuclear Information System (INIS)

Holzer, H.; Purwin, C.; Pohlig, G.; Scheffers, W.A.; Nicolay, K.

1986-01-01

Addition of fermentable sugars or uncouplers such as CCCP to resting yeast cells grown on glucose initiates phosphorylation of fructose-1,6-bisphosphatase (FBPase). There is good evidence that phosphorylation marks FBPase for proteolytic degradation. 31 P-NMR measurements of the cytosolic pH of yeast cells demonstrated a decrease of the cytosolic pH from 7.0 to 6.5 after addition of glucose or CCCP to starved yeast. Activity of adenylate cyclase in permeabilized yeast cells increases 2-3-fold when the pH is lowered from 7.0 to 6.5. It is concluded that pH controlled activation of adenylate cyclase causes the previously described increase in cyclic AMP which leads to phosphorylation of FBPase and finally to proteolysis of FBPase

Akt-dependent Activation of the Heart 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFKFB2) Isoenzyme by Amino Acids*

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Novellasdemunt, Laura; Tato, Irantzu; Navarro-Sabate, Aurea; Ruiz-Meana, Marisol; Méndez-Lucas, Andrés; Perales, Jose Carlos; Garcia-Dorado, David; Ventura, Francesc; Bartrons, Ramon; Rosa, Jose Luis

2013-01-01

Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked. PMID:23457334

Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

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Barretto O.C. de O.

2006-01-01

Full Text Available In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa. The Michaelis-Menten constants (Km: 55 µM for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively. A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

Subtilisin-catalyzed esterification of di- and oligosaccharides containing a d-fructose moiety

International Nuclear Information System (INIS)

Riva, S.; Nonini, M.; Ottolina, G.; Danieli, B.

1998-01-01

Several di- and oligosaccharides containing a d-fructose moiety have been acylated by protease subtilisin in anhydrous dimethylformamide in the presence of the activated ester trifluoroethyl butanoate. Under the reaction conditions used, all the substrates were converted into the corresponding monobutanoates in ca. 50% isolated yields. Structural determination of the products by 13 C NMR indicated a strong preference of subtilisin towards the regioselective esterification of the primary hydroxyls of the fructose moiety and, specifically, of the C-1 OH, as already observed with sucrose. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

SLC37A1 and SLC37A2 are phosphate-linked, glucose-6-phosphate antiporters.

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Chi-Jiunn Pan

Full Text Available Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P into glucose and phosphate (P(i. This reaction depends on coupling the G6P transporter (G6PT with glucose-6-phosphatase-α (G6Pase-α. Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.

High-Fructose Corn-Syrup-Sweetened Beverage Intake Increases 5-Hour Breast Milk Fructose Concentrations in Lactating Women.

Science.gov (United States)

Berger, Paige K; Fields, David A; Demerath, Ellen W; Fujiwara, Hideji; Goran, Michael I

2018-05-24

This study determined the effects of consuming a high-fructose corn syrup (HFCS)-sweetened beverage on breast milk fructose, glucose, and lactose concentrations in lactating women. At six weeks postpartum, lactating mothers ( n = 41) were randomized to a crossover study to consume a commercially available HFCS-sweetened beverage or artificially sweetened control beverage. At each session, mothers pumped a complete breast milk expression every hour for six consecutive hours. The baseline fasting concentrations of breast milk fructose, glucose, and lactose were 5.0 ± 1.3 µg/mL, 0.6 ± 0.3 mg/mL, and 6.8 ± 1.6 g/dL, respectively. The changes over time in breast milk sugars were significant only for fructose (treatment × time, p fructose at 120 min (8.8 ± 2.1 vs. 5.3 ± 1.9 µg/mL), 180 min (9.4 ± 1.9 vs. 5.2 ± 2.2 µg/mL), 240 min (7.8 ± 1.7 vs. 5.1 ± 1.9 µg/mL), and 300 min (6.9 ± 1.4 vs. 4.9 ± 1.9 µg/mL) (all p fructose was also different between treatments (14.7 ± 1.2 vs. -2.60 ± 1.2 µg/mL × 360 min, p glucose or lactose. Our data suggest that the consumption of an HFCS-sweetened beverage increased breast milk fructose concentrations, which remained elevated up to five hours post-consumption.

Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis.

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Oh, Euhlim; Lu, Mingshou; Park, Changhun; Park, Changhun; Oh, Han Bin; Lee, Sang Yup; Lee, Jinwon

2011-02-01

A dynamic model of lactic acid fermentation using Lactococcus lactis was constructed, and a metabolic flux analysis (MFA) and metabolic control analysis (MCA) were performed to reveal an intensive metabolic understanding of lactic acid bacteria (LAB). The parameter estimation was conducted with COPASI software to construct a more accurate metabolic model. The experimental data used in the parameter estimation were obtained from an LC-MS/ MS analysis and time-course simulation study. The MFA results were a reasonable explanation of the experimental data. Through the parameter estimation, the metabolic system of lactic acid bacteria can be thoroughly understood through comparisons with the original parameters. The coefficients derived from the MCA indicated that the reaction rate of L-lactate dehydrogenase was activated by fructose 1,6-bisphosphate and pyruvate, and pyruvate appeared to be a stronger activator of L-lactate dehydrogenase than fructose 1,6-bisphosphate. Additionally, pyruvate acted as an inhibitor to pyruvate kinase and the phosphotransferase system. Glucose 6-phosphate and phosphoenolpyruvate showed activation effects on pyruvate kinase. Hexose transporter was the strongest effector on the flux through L-lactate dehydrogenase. The concentration control coefficient (CCC) showed similar results to the flux control coefficient (FCC).

Real-time monitoring of sucrose, sorbitol, d-glucose and d-fructose concentration by electromagnetic sensing.

Science.gov (United States)

Harnsoongnoen, Supakorn; Wanthong, Anuwat

2017-10-01

Magnetic sensing at microwave frequencies for real-time monitoring of sucrose, sorbitol, d-glucose and d-fructose concentrations is reported. The sensing element was designed based on a coplanar waveguide (CPW) loaded with a split ring resonator (SRR), which was fabricated on a DiClad 880 substrate with a thickness of 1.6mm and relative permittivity (ε r ) of 2.2. The magnetic sensor was connected to a Vector Network Analyzer (VNA) and the electromagnetic interaction between the samples and sensor was analyzed. The magnitude of the transmission coefficient (S 21 ) was used as an indicator to detect the solution sample concentrations ranging from 0.04 to 0.20g/ml. The experimental results confirmed that the developed system using microwaves for the real-time monitoring of sucrose, sorbitol, d-glucose and d-fructose concentrations gave unique results for each solution type and concentration. Moreover, the proposed sensor has a wide dynamic range, high linearity, fast operation and low-cost. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Relationship between Reduced Nucleus Accumbens Shell and Enhanced Lateral Hypothalamic Orexin Neuronal Activation in Long-Term Fructose Bingeing Behavior

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Rorabaugh, Jacki M.; Stratford, Jennifer M.; Zahniser, Nancy R.

2014-01-01

Fructose accounts for 10% of daily calories in the American diet. Fructose, but not glucose, given intracerebroventricularly stimulates homeostatic feeding mechanisms within the hypothalamus; however, little is known about how fructose affects hedonic feeding centers. Repeated ingestion of sucrose, a disaccharide of fructose and glucose, increases neuronal activity in hedonic centers, the nucleus accumbens (NAc) shell and core, but not the hypothalamus. Rats given glucose in the intermittent access model (IAM) display signatures of hedonic feeding including bingeing and altered DA receptor (R) numbers within the NAc. Here we examined whether substituting fructose for glucose in this IAM produces bingeing behavior, alters DA Rs and activates hedonic and homeostatic feeding centers. Following long-term (21-day) exposure to the IAM, rats given 8–12% fructose solutions displayed fructose bingeing but unaltered DA D1R or D2R number. Fructose bingeing rats, as compared to chow bingeing controls, exhibited reduced NAc shell neuron activation, as determined by c-Fos-immunoreactivity (Fos-IR). This activation was negatively correlated with orexin (Orx) neuron activation in the lateral hypothalamus/perifornical area (LH/PeF), a brain region linking homeostatic to hedonic feeding centers. Following short-term (2-day) access to the IAM, rats exhibited bingeing but unchanged Fos-IR, suggesting only long-term fructose bingeing increases Orx release. In long-term fructose bingeing rats, pretreatment with the Ox1R antagonist SB-334867 (30 mg/kg; i.p.) equally reduced fructose bingeing and chow intake, resulting in a 50% reduction in calories. Similarly, in control rats, SB-334867 reduced chow/caloric intake by 60%. Thus, in the IAM, Ox1Rs appear to regulate feeding based on caloric content rather than palatability. Overall, our results, in combination with the literature, suggest individual monosaccharides activate distinct neuronal circuits to promote feeding behavior

A relationship between reduced nucleus accumbens shell and enhanced lateral hypothalamic orexin neuronal activation in long-term fructose bingeing behavior.

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Jacki M Rorabaugh

Full Text Available Fructose accounts for 10% of daily calories in the American diet. Fructose, but not glucose, given intracerebroventricularly stimulates homeostatic feeding mechanisms within the hypothalamus; however, little is known about how fructose affects hedonic feeding centers. Repeated ingestion of sucrose, a disaccharide of fructose and glucose, increases neuronal activity in hedonic centers, the nucleus accumbens (NAc shell and core, but not the hypothalamus. Rats given glucose in the intermittent access model (IAM display signatures of hedonic feeding including bingeing and altered DA receptor (R numbers within the NAc. Here we examined whether substituting fructose for glucose in this IAM produces bingeing behavior, alters DA Rs and activates hedonic and homeostatic feeding centers. Following long-term (21-day exposure to the IAM, rats given 8-12% fructose solutions displayed fructose bingeing but unaltered DA D1R or D2R number. Fructose bingeing rats, as compared to chow bingeing controls, exhibited reduced NAc shell neuron activation, as determined by c-Fos-immunoreactivity (Fos-IR. This activation was negatively correlated with orexin (Orx neuron activation in the lateral hypothalamus/perifornical area (LH/PeF, a brain region linking homeostatic to hedonic feeding centers. Following short-term (2-day access to the IAM, rats exhibited bingeing but unchanged Fos-IR, suggesting only long-term fructose bingeing increases Orx release. In long-term fructose bingeing rats, pretreatment with the Ox1R antagonist SB-334867 (30 mg/kg; i.p. equally reduced fructose bingeing and chow intake, resulting in a 50% reduction in calories. Similarly, in control rats, SB-334867 reduced chow/caloric intake by 60%. Thus, in the IAM, Ox1Rs appear to regulate feeding based on caloric content rather than palatability. Overall, our results, in combination with the literature, suggest individual monosaccharides activate distinct neuronal circuits to promote feeding behavior

Hereditary fructose intolerance

Science.gov (United States)

Fructosemia; Fructose intolerance; Fructose aldolase B-deficiency; Fructose-1, 6-bisphosphate aldolase deficiency ... B. This substance is needed to break down fructose. If a person without this substance eats fructose ...

Bisulphite complexation in the resin catalysed epimerisation of D-fructose-radiotracer investigations [Paper No. AR-27

International Nuclear Information System (INIS)

Murlidharan, B.; Mallika, S.; Viswanathan, K.V.

1982-01-01

Tracer methods were used to study the conversion of D-Fructose- 16 C to its aldose epimers. D-Fructose- 14 C sorbed on Dowex-1-X8(OH - form) columns on elution with dilute sulphurous acid (>0.1M) was converted mainly to D-Glucose- 14 C accompanied by excessive degradation. Treatment of D-Fructose- 14 C with Dowex-1-X4 in carbonate and bisulphite forms at 50degC in 50 per cent ethanol gave an epimeric mixture containing more than 25 per cent of D-Mannose- 14 C. (author)

Signaling Role of Fructose Mediated by FINS1/FBP in Arabidopsis thaliana

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Cho, Young-Hee; Yoo, Sang-Dong

2011-01-01

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1–dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1–dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination. PMID:21253566

Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

LENUS (Irish Health Repository)

Semjonous, Nina M

2011-01-01

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1\\/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)\\/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

SwissProt search result: AK107595 [KOME

Lifescience Database Archive (English)

Full Text Available AK107595 002-131-A11 (Q74GH6) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_GEOSL 1e-11 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q74GH6) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_GEOSL 1e-59 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (O19908) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CYACA 1e-46 ...

Short-term dietary supplementation with fructose accelerates gastric emptying of a fructose but not a glucose solution.

Science.gov (United States)

Yau, Adora M W; McLaughlin, John; Maughan, Ronald J; Gilmore, William; Evans, Gethin H

2014-01-01

Short-term dietary glucose supplementation has been shown to accelerate the gastric emptying rate of both glucose and fructose solutions. The aim of this study was to examine gastric emptying rate responses to monosaccharide ingestion following short-term dietary fructose supplementation. The gastric emptying rate of a fructose solution containing 36 g of fructose and an equicaloric glucose solution containing 39.6 g glucose monohydrate were measured in 10 healthy non-smoking men with and without prior fructose supplementation (water control) using a randomized crossover design. Gastric emptying rate was assessed for a period of 1 h using the [(13)C]breath test with sample collections at baseline and 10-min intervals following drink ingestion. Additionally, appetite ratings of hunger, fullness, and prospective food consumption were recorded at baseline and every 10 min using visual analog scales. Increased dietary fructose ingestion resulted in significantly accelerated half-emptying time of a fructose solution (mean = 48, SD = 6 versus 58, SD = 14 min control; P = 0.037), whereas the emptying of a glucose solution remained unchanged (mean = 85, SD = 31 versus 78, SD = 27 min control; P = 0.273). Time of maximal emptying rate of fructose was also significantly accelerated following increased dietary fructose intake (mean = 33, SD = 6 versus 38, SD = 9 min control; P = 0.042), while it remained unchanged for glucose (mean = 45, SD = 14 versus 44, SD = 14 min control; P = 0.757). No effects of supplementation were observed for appetite measures. Three d of supplementation with 120 g/d of fructose resulted in an acceleration of gastric emptying rate of a fructose solution but not a glucose solution. Copyright © 2014 Elsevier Inc. All rights reserved.

The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

Directory of Open Access Journals (Sweden)

Sergio Zonszein

Full Text Available The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P deaminase from Escherichia coli (EcGNPDA as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P. We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with Kt and Kr as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q9Z6U0) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CHLPN 1e-36 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q7VRZ3) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_BORPE 1e-52 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q9ZJ94) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_HELPJ 1e-31 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q8Y915) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_LISMO 1e-41 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q8KA75) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_BUCAP 1e-96 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q5HM69) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_STAEQ 1e-40 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q97SQ9) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_STRPN 1e-53 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q890U2) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CLOTE 1e-50 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q663R1) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_YERPS 2e-56 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q821Z7) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CHLCV 1e-39 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q88YE7) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_LACPL 1e-47 ...

Allosteric regulation of 6-phosphofructo-1-kinase activity of fat body and flight muscle from the bloodsucking bug Rhodnius prolixus

Directory of Open Access Journals (Sweden)

Gutemberg G. Alves

2007-03-01

Full Text Available 6-phosphofructo-1-kinase (phosphofructokinase; PFK activity from Rhodnius prolixus, a haematophagous insect which is usually a poor flyer, was measured and compared in two metabolically active tissues - flight muscle and fat body. The activity of this important regulatory glycolytic enzyme was much more pronounced in muscle (15.1 ± 1.4 U/mg than in fat body extracts (3.6±0.4 U/mg, although the latter presented higher levels of enzyme per protein content, as measured by western-blotting. Muscle extracts are more responsible than fat body to ATP and fructose 6-phosphate, both substrates of PFK. Allosteric regulation exerted by different effectors such as ADP, AMP and fructose 2,6-phosphate presented a singular pattern for each tissue. Optimal pH (8.0-8.5 and sensitivity to pH variation was very similar, and citrate was unable to inhibit PFK activity in both extracts. Our results suggest the existence of a particular PFK activity for each tissue, with regulatory patterns that are consistent with their physiological roles.A atividade da fosfofrutocinase (PFK de Rodnius prolixus, um inseto hematófago, o qual vôa somente pequenas distâncias, foi medida e comparada em dois tecidos metabolicamente ativos - músculo de asa e corpo gorduroso. A atividade desta importante enzima glicolítica regulatória foi muito mais pronunciada em músculo de asa (15,1 ±1,4 U/mg do que em extrato de corpo gorduroso (3,6 ±0,4 U/mg embora este último tenha apresentado níveis mais altos da enzima por quantidade de proteína, como medido por western-blotting. Extratos de músculo foram mais responsivos do que corpo gorduroso para ATP e frutose-6-fosfato, ambos substratos da PFK. A regulação alostérica exercida por diferentes efetores tais como ADP, AMP, frutose-2,6-bisfosfato apresentou um padrão singular para cada tecido. O pH ótimo (8,0-8,5 e a sensibilidade a variações de pH, foram muito similares e o citrato foi incapaz de inibir a atividade da PFK em

Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size, but only small changes in carbohydrate metabolism

DEFF Research Database (Denmark)

Obiadalla-Ali, H.; Fernie, A.R.; Lytovchenko, A.

2004-01-01

A potato (Solanum tuberosum L. ) cDNA coding for the chloroplastic isoform of fructose 1,6-bisphosphatase (cp-FBPase) was utilized to repress its activity in tomatoes (Lycopersicon esculentum Mill.) using antisense techniques. The patatin B33 promoter was used to ensure fruit specificity of the a...

D-Tagatose inhibits the growth and biofilm formation of Streptococcus mutans

Science.gov (United States)

Hasibul, Khaleque; Nakayama-Imaohji, Haruyuki; Hashimoto, Masahito; Yamasaki, Hisashi; Ogawa, Takaaki; Waki, Junpei; Tada, Ayano; Yoneda, Saori; Tokuda, Masaaki; Miyake, Minoru; Kuwahara, Tomomi

2018-01-01

Dental caries is an important global health concern and Streptococcus mutans has been established as a major cariogenic bacterial species. Reports indicate that a rare sugar, D-tagatose, is not easily catabolized by pathogenic bacteria. In the present study, the inhibitory effects of D-tagatose on the growth and biofilm formation of S. mutans GS-5 were examined. Monitoring S. mutans growth over a 24 h period revealed that D-tagatose prolonged the lag phase without interfering with the final cell yield. This growth retardation was also observed in the presence of 1% sucrose, although it was abolished by the addition of D-fructose. S. mutans biofilm formation was significantly inhibited by growth in sucrose media supplemented with 1 and 4% D-tagatose compared with that in a culture containing sucrose alone, while S. mutans formed granular biofilms in the presence of this rare sugar. The inhibitory effect of D-tagatose on S. mutans biofilm formation was significantly more evident than that of xylitol. Growth in sucrose media supplemented with D-tagatose significantly decreased the expression of glucosyltransferase, exo-β-fructosidase and D-fructose-specific phosphotransferase genes but not the expression of fructosyltransferase compared with the culture containing sucrose only. The activity of cell-associated glucosyltransferase in S. mutans was inhibited by 4% D-tagatose. These results indicate that D-tagatose reduces water-insoluble glucan production from sucrose by inhibiting glucosyltransferase activities, which limits access to the free D-fructose released during this process and retards the growth of S. mutans. Therefore, foods and oral care products containing D-tagatose are anticipated to reduce the risk of caries by inhibiting S. mutans biofilm formation. PMID:29115611

d-Fructose-Decorated Poly(ethylene imine) for Human Breast Cancer Cell Targeting.

Science.gov (United States)

Englert, Christoph; Pröhl, Michael; Czaplewska, Justyna A; Fritzsche, Carolin; Preußger, Elisabeth; Schubert, Ulrich S; Traeger, Anja; Gottschaldt, Michael

2017-08-01

The high affinity of GLUT5 transporter for d-fructose in breast cancer cells has been discussed intensely. In this contribution, high molar mass linear poly(ethylene imine) (LPEI) is functionalized with d-fructose moieties to combine the selectivity for the GLUT5 transporter with the delivery potential of PEI for genetic material. The four-step synthesis of a thiol-group bearing d-fructose enables the decoration of a cationic polymer backbone with d-fructose via thiol-ene photoaddition. The functionalization of LPEI is confirmed by 2D NMR techniques, elemental analysis, and size exclusion chromatography. Importantly, a d-fructose decoration of 16% renders the polymers water-soluble and eliminates the cytotoxicity of PEI in noncancer L929 cells, accompanied by a reduced unspecific cellular uptake of the genetic material. In contrast, the cytotoxicity as well as the cell specific uptake is increased for triple negative MDA-MB-231 breast cancer cells. Therefore, the introduction of d-fructose shows superior potential for cell targeting, which can be assumed to be GLUT5 dependent. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet.

Directory of Open Access Journals (Sweden)

Komal Sodhi

Full Text Available Oxidative stress underlies the etiopathogenesis of nonalcoholic fatty liver disease (NAFLD, obesity and cardiovascular disease (CVD. Heme Oxygenase-1 (HO-1 is a potent endogenous antioxidant gene that plays a key role in decreasing oxidative stress. Sirtuin1 (SIRT1 belongs to the family of NAD-dependent de-acyetylases and is modulated by cellular redox.We hypothesize that fructose-induced obesity creates an inflammatory and oxidative environment conducive to the development of NAFLD and metabolic syndrome. The aim of this study is to determine whether HO-1 acts through SIRT1 to form a functional module within hepatocytes to attenuate steatohepatitis, hepatic fibrosis and cardiovascular dysfunction.We examined the effect of fructose, on hepatocyte lipid accumulation and fibrosis in murine hepatocytes and in mice fed a high fructose diet in the presence and absence of CoPP, an inducer of HO-1, and SnMP, an inhibitor of HO activity. Fructose increased oxidative stress markers and decreased HO-1 and SIRT1 levels in hepatocytes (p<0.05. Further fructose supplementation increased FAS, PPARα, pAMPK and triglycerides levels; CoPP negated this increase. Concurrent treatment with CoPP and SIRT1 siRNA in hepatocytes increased FAS, PPARα, pAMPK and triglycerides levels suggesting that HO-1 is upstream of SIRT1 and suppression of SIRT1 attenuates the beneficial effects of HO-1. A high fructose diet increased insulin resistance, blood pressure, markers of oxidative stress and lipogenesis along with fibrotic markers in mice (p<0.05. Increased levels of HO-1 increased SIRT1 levels and ameliorated fructose-mediated lipid accumulation and fibrosis in liver along with decreasing vascular dysfunction (p<0.05 vs. fructose. These beneficial effects of CoPP were reversed by SnMP.Taken together, our study demonstrates, for the first time, that HO-1 induction attenuates fructose-induced hepatic lipid deposition, prevents the development of hepatic fibrosis and abates

High d(+)-fructose diet adversely affects testicular weight gain in weaning rats─protection by moderate d(+)-glucose diet.

Science.gov (United States)

Shibata, Katsumi; Fukuwatari, Tsutomu

2013-01-01

The use of high D(+)-fructose corn syrup has increased over the past several decades in the developed countries, while overweight and obesity rates and the related diseases have risen dramatically. However, we found that feeding a high D(+)-fructose diet (80% D(+)-fructose as part of the diet) to weaning rats for 21 days led to reduced food intake (50% less, P fructose diet. We also challenged a minimum requirement of dietary D(+)-glucose for preventing the adverse effects of D(+)-fructose, such as lower food intake and reduction of body weight and testicular weight; the minimum requirement of D(+)-glucose was ≈23% of the diet. This glucose amount may be the minimum requirement of exogenous glucose for reducing weight gain.

High-Fructose Corn-Syrup-Sweetened Beverage Intake Increases 5-Hour Breast Milk Fructose Concentrations in Lactating Women

Directory of Open Access Journals (Sweden)

Paige K. Berger

2018-05-01

Full Text Available This study determined the effects of consuming a high-fructose corn syrup (HFCS-sweetened beverage on breast milk fructose, glucose, and lactose concentrations in lactating women. At six weeks postpartum, lactating mothers (n = 41 were randomized to a crossover study to consume a commercially available HFCS-sweetened beverage or artificially sweetened control beverage. At each session, mothers pumped a complete breast milk expression every hour for six consecutive hours. The baseline fasting concentrations of breast milk fructose, glucose, and lactose were 5.0 ± 1.3 µg/mL, 0.6 ± 0.3 mg/mL, and 6.8 ± 1.6 g/dL, respectively. The changes over time in breast milk sugars were significant only for fructose (treatment × time, p < 0.01. Post hoc comparisons showed the HFCS-sweetened beverage vs. control beverage increased breast milk fructose at 120 min (8.8 ± 2.1 vs. 5.3 ± 1.9 µg/mL, 180 min (9.4 ± 1.9 vs. 5.2 ± 2.2 µg/mL, 240 min (7.8 ± 1.7 vs. 5.1 ± 1.9 µg/mL, and 300 min (6.9 ± 1.4 vs. 4.9 ± 1.9 µg/mL (all p < 0.05. The mean incremental area under the curve for breast milk fructose was also different between treatments (14.7 ± 1.2 vs. −2.60 ± 1.2 µg/mL × 360 min, p < 0.01. There was no treatment × time interaction for breast milk glucose or lactose. Our data suggest that the consumption of an HFCS-sweetened beverage increased breast milk fructose concentrations, which remained elevated up to five hours post-consumption.

Beneficial effects of calcitriol on hypertension, glucose intolerance, impairment of endothelium-dependent vascular relaxation, and visceral adiposity in fructose-fed hypertensive rats.

Science.gov (United States)

Chou, Chu-Lin; Pang, Cheng-Yoong; Lee, Tony J F; Fang, Te-Chao

2015-01-01

Besides regulating calcium homeostasis, the effects of vitamin D on vascular tone and metabolic disturbances remain scarce in the literature despite an increase intake with high-fructose corn syrup worldwide. We investigated the effects of calcitriol, an active form of vitamin D, on vascular relaxation, glucose tolerance, and visceral fat pads in fructose-fed rats. Male Wistar-Kyoto rats were divided into 4 groups (n = 6 per group). Group Con: standard chow diet for 8 weeks; Group Fru: high-fructose diet (60% fructose) for 8 weeks; Group Fru-HVD: high-fructose diet as Group Fru, high-dose calcitriol treatment (20 ng / 100 g body weight per day) 4 weeks after the beginning of fructose feeding; and Group Fru-LVD: high-fructose diet as Group Fru, low-dose calcitriol treatment (10 ng / 100 g body weight per day) 4 weeks after the beginning of fructose feeding. Systolic blood pressure was measured twice a week by the tail-cuff method. Blood was examined for serum ionized calcium, phosphate, creatinine, glucose, triglycerides, and total cholesterol. Intra-peritoneal glucose intolerance test, aortic vascular reactivity, the weight of visceral fat pads, adipose size, and adipose angiotensin II levels were analyzed at the end of the study. The results showed that the fructose-fed rats significantly developed hypertension, impaired glucose tolerance, heavier weight and larger adipose size of visceral fat pads, and raised adipose angiotensin II expressions compared with the control rats. High- and low-dose calcitriol reduced modestly systolic blood pressure, increased endothelium-dependent aortic relaxation, ameliorated glucose intolerance, reduced the weight and adipose size of visceral fat pads, and lowered adipose angiotensin II expressions in the fructose-fed rats. However, high-dose calcitriol treatment mildly increased serum ionized calcium levels (1.44 ± 0.05 mmol/L). These results suggest a protective role of calcitriol treatment on endothelial function, glucose

Beneficial effects of calcitriol on hypertension, glucose intolerance, impairment of endothelium-dependent vascular relaxation, and visceral adiposity in fructose-fed hypertensive rats.

Directory of Open Access Journals (Sweden)

Chu-Lin Chou

Full Text Available Besides regulating calcium homeostasis, the effects of vitamin D on vascular tone and metabolic disturbances remain scarce in the literature despite an increase intake with high-fructose corn syrup worldwide. We investigated the effects of calcitriol, an active form of vitamin D, on vascular relaxation, glucose tolerance, and visceral fat pads in fructose-fed rats. Male Wistar-Kyoto rats were divided into 4 groups (n = 6 per group. Group Con: standard chow diet for 8 weeks; Group Fru: high-fructose diet (60% fructose for 8 weeks; Group Fru-HVD: high-fructose diet as Group Fru, high-dose calcitriol treatment (20 ng / 100 g body weight per day 4 weeks after the beginning of fructose feeding; and Group Fru-LVD: high-fructose diet as Group Fru, low-dose calcitriol treatment (10 ng / 100 g body weight per day 4 weeks after the beginning of fructose feeding. Systolic blood pressure was measured twice a week by the tail-cuff method. Blood was examined for serum ionized calcium, phosphate, creatinine, glucose, triglycerides, and total cholesterol. Intra-peritoneal glucose intolerance test, aortic vascular reactivity, the weight of visceral fat pads, adipose size, and adipose angiotensin II levels were analyzed at the end of the study. The results showed that the fructose-fed rats significantly developed hypertension, impaired glucose tolerance, heavier weight and larger adipose size of visceral fat pads, and raised adipose angiotensin II expressions compared with the control rats. High- and low-dose calcitriol reduced modestly systolic blood pressure, increased endothelium-dependent aortic relaxation, ameliorated glucose intolerance, reduced the weight and adipose size of visceral fat pads, and lowered adipose angiotensin II expressions in the fructose-fed rats. However, high-dose calcitriol treatment mildly increased serum ionized calcium levels (1.44 ± 0.05 mmol/L. These results suggest a protective role of calcitriol treatment on endothelial

Genetics Home Reference: glucose phosphate isomerase deficiency

Science.gov (United States)

... glycolytic pathway; in this step, a molecule called glucose-6-phosphate is converted to another molecule called fructose-6-phosphate. When GPI remains a single molecule (a monomer) it is involved in the development and maintenance of nerve cells ( neurons ). In this context, it is often known as ...

Dietary phenolic acids reverse insulin resistance, hyperglycaemia, dyslipidaemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome rats.

Science.gov (United States)

Ibitoye, Oluwayemisi B; Ajiboye, Taofeek O

2017-12-20

This study investigated the influence of caffeic, ferulic, gallic and protocatechuic acids on high-fructose diet-induced metabolic syndrome in rats. Oral administration of the phenolic acids significantly reversed high-fructose diet-mediated increase in body mass index and blood glucose. Furthermore, phenolic acids restored high-fructose diet-mediated alterations in metabolic hormones (insulin, leptin and adiponectin). Similarly, elevated tumour necrosis factor-α, interleukin-6 and -8 were significantly lowered. Administration of phenolic acids restored High-fructose diet-mediated increase in the levels of lipid parameters and indices of atherosclerosis, cardiac and cardiovascular diseases. High-fructose diet-mediated decrease in activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase) and increase in oxidative stress biomarkers (reduced glutathione, lipid peroxidation products, protein oxidation and fragmented DNA) were significantly restored by the phenolic acids. The result of this study shows protective influence of caffeic acid, ferulic acid, gallic acid and protocatechuic acid in high-fructose diet-induced metabolic syndrome.

The Effect of D-Tagatose on Fructose Absorption in a Rat Model

OpenAIRE

Williams, Jarrod; Spitnale, Michael; Lodder, Robert

2013-01-01

D-tagatose is in development as a medication for the treatment of type 2 diabetes. The effect of oral D-tagatose on the absorption of D-fructose was assessed when co-administered in this study. In the pilot study, male Sprague-Dawley rats were fed C14 labeled fructose and glucose concomitantly to establish dose levels for the treatment group of rats fed C14 labeled fructose together with D-tagatose. Rats were administered 0, 600, 2000, 6000, or 12000 mg/kg of D-tagatose along with 2000 mg/kg ...

Interaction between D-fructose dehydrogenase and methoxy-substituent-functionalized carbon surface to increase productive orientations

International Nuclear Information System (INIS)

Xia, Hong-qi; Hibino, Yuya; Kitazumi, Yuki; Shirai, Osamu; Kano, Kenji

2016-01-01

Highlights: • Methoxy-functionalized surface improves the DET-type bioelectrocatalysis of FDH. • Methoxy-functionalized surface increases productive orientations. • The total catalytic activity of FDH is almost independent of the modification. • High current density as well as good stability is useful for biofuel cells. - Abstract: D-Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 catalyzes the two-electron oxidation of D-fructose to 5-keto-D-fructose, and it is widely used in biofuel cells and biosensors. In this study, methoxy-substituent-functionalized carbon electrodes are constructed by electrochemical oxidation of methoxy-aniline derivatives on Ketjen Black (KB)-modified electrodes to improve the immobilization and bioelectrocatalysis of FDH. It is proposed that the specific interaction between FDH, especially the heme c moiety, and methoxy substituent(s) of amines on carbon electrode increases the proportion of the productively oriented FDH molecules to the total FDHs. Consequently, the limiting catalytic current density of the D-fructose oxidation increases to as much as 23 ± 2 mA cm −2 in FDH/2,4-dimethoxyaniline/KB/glassy carbon electrode, for example.

An homolog of the Frz Phosphoenolpyruvate:carbohydrate phosphoTransferase System of extraintestinal pathogenic Escherichia coli is encoded on a genomic island in specific lineages of Streptococcus agalactiae.

Science.gov (United States)

Patron, Kévin; Gilot, Philippe; Camiade, Emilie; Mereghetti, Laurent

2015-06-01

We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults. Copyright © 2015 Elsevier B.V. All rights reserved.

High D(+-Fructose Diet Adversely Affects Testicular Weight Gain in Weaning Rats—Protection by Moderate D(+-Glucose Diet

Directory of Open Access Journals (Sweden)

Katsumi Shibata

2013-01-01

Full Text Available The use of high D(+-fructose corn syrup has increased over the past several decades in the developed countries, while overweight and obesity rates and the related diseases have risen dramatically. However, we found that feeding a high D(+-fructose diet (80% D(+-fructose as part of the diet to weaning rats for 21 days led to reduced food intake (50% less, P < 0.0001 and thus delayed the weight gains in the body (40% less, P < 0.0001 and testes (40% less, P < 0.0001 compared to the no D(+-fructose diet. We also challenged a minimum requirement of dietary D(+-glucose for preventing the adverse effects of D(+-fructose, such as lower food intake and reduction of body weight and testicular weight; the minimum requirement of D(+-glucose was ã23% of the diet. This glucose amount may be the minimum requirement of exogenous glucose for reducing weight gain.

Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

OpenAIRE

Maruta, Takanori; Yonemitsu, Miki; Yabuta, Yukinori; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

2008-01-01

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 ...

Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system

DEFF Research Database (Denmark)

Rohwer, J.M.; Jensen, Peter Ruhdal; Shinohara, Y.

1996-01-01

The effect of different cellular free-energy states on the uptake of methyl alfa-D-glucopyranoside, an analoque of glucose, by Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular ATP/ADP ratio was varied by changing the expression...... of the atp operon, which codes for the H+-ATPase, or by adding an uncoupler of oxidative phosphorylation or an inhibitor of respiration. Corresponding initial phosphotransferase uptake rates were determined using an improved uptake assay that works with growing cells in steady state. The results show...... that the initial uptake rate was decreased under conditions of lowered intracellular ATP/ADP ratios, irrespective of which method was used to change the cellular energy state.. When either the expression of the atp operon was changed or 2,4-dinitrophenol was added to wild-type cells, the relationship between...

Fructose effect to enhance liver glycogen deposition is due to inhibition of glycogenolysis

International Nuclear Information System (INIS)

Youn, J.; Kaslow, H.; Bergman, R.

1987-01-01

The effect of fructose on glycogen degradation was examined by measuring flux of [ 14 C] from prelabeled glycogen in perfused rat livers. During 2 h refeeding of fasted rats hepatic glycogen was labeled by injection of [U 14 C] galactose (0.1 mg and 0.02 μCi/g of body weight). Refed livers were perfused for 30 min with glucose only (10 mM) and for 60 min with glucose (10 mM) without (n=5) or with fructose (1, 2, 10 mM; n=5 for each). With fructose, label production immediately declined and remained suppressed through the end of perfusion (P < 0.05). Suppression was dose-dependent: steady state label production was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose (P < 0.0001), without significant changes in glycogen synthase or phosphorylase. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (F1P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.6 μmoles/g of liver with 1, 2, and 10 mM fructose. Maximum inhibition of phosphorylase was 82%; FIP concentration for half inhibition was 0.57 μmoles/g of liver, well within the concentration of F1P attained in refeeding. Fructose enhances net glycogen synthesis in liver by suppressing glycogenolysis and the suppression is presumably caused by allosteric inhibition of phosphorylase by F1P

Fructose suppresses uric acid excretion to the intestinal lumen as a result of the induction of oxidative stress by NADPH oxidase activation.

Science.gov (United States)

Kaneko, Chihiro; Ogura, Jiro; Sasaki, Shunichi; Okamoto, Keisuke; Kobayashi, Masaki; Kuwayama, Kaori; Narumi, Katsuya; Iseki, Ken

2017-03-01

A high intake of fructose increases the risk for hyperuricemia. It has been reported that long-term fructose consumption suppressed renal uric acid excretion and increased serum uric acid level. However, the effect of single administration of fructose on excretion of uric acid has not been clarified. We used male Wistar rats, which were orally administered fructose (5g/kg). Those rats were used in each experiment at 12h after administration. Single administration of fructose suppressed the function of ileal uric acid excretion and had no effect on the function of renal uric acid excretion. Breast cancer resistance protein (BCRP) predominantly contributes to intestinal excretion of uric acid as an active homodimer. Single administration of fructose decreased BCRP homodimer level in the ileum. Moreover, diphenyleneiodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), recovered the suppression of the function of ileal uric acid excretion and the Bcrp homodimer level in the ileum of rats that received single administration of fructose. Single administration of fructose decreases in BCRP homodimer level, resulting in the suppression the function of ileal uric acid excretion. The suppression of the function of ileal uric acid excretion by single administration of fructose is caused by the activation of Nox. The results of our study provide a new insight into the mechanism of fructose-induced hyperuricemia. Copyright © 2016 Elsevier B.V. All rights reserved.

Uric Acid Stimulates Fructokinase and Accelerates Fructose Metabolism in the Development of Fatty Liver

Science.gov (United States)

Lanaspa, Miguel A.; Sanchez-Lozada, Laura G.; Cicerchi, Christina; Li, Nanxing; Roncal-Jimenez, Carlos A.; Ishimoto, Takuji; Le, Myphuong; Garcia, Gabriela E.; Thomas, Jeffrey B.; Rivard, Christopher J.; Andres-Hernando, Ana; Hunter, Brandi; Schreiner, George; Rodriguez-Iturbe, Bernardo; Sautin, Yuri Y.; Johnson, Richard J.

2012-01-01

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver. PMID:23112875

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

Science.gov (United States)

Van Schaftingen, E; Vandercammen, A

1989-01-15

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.

Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides : The coupling between transport and phosphorylation in inside-out vesicles

NARCIS (Netherlands)

Lolkema, Juke S.; Robillard, George T.

The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the

Involvement of glucocorticoid prereceptor metabolism and signaling in rat visceral adipose tissue lipid metabolism after chronic stress combined with high-fructose diet.

Science.gov (United States)

Bursać, Biljana; Djordjevic, Ana; Veličković, Nataša; Milutinović, Danijela Vojnović; Petrović, Snježana; Teofilović, Ana; Gligorovska, Ljupka; Preitner, Frederic; Tappy, Luc; Matić, Gordana

2018-05-03

Both fructose overconsumption and increased glucocorticoids secondary to chronic stress may contribute to overall dyslipidemia. In this study we specifically assessed the effects and interactions of dietary fructose and chronic stress on lipid metabolism in the visceral adipose tissue (VAT) of male Wistar rats. We analyzed the effects of 9-week 20% high fructose diet and 4-week chronic unpredictable stress, separately and in combination, on VAT histology, glucocorticoid prereceptor metabolism, glucocorticoid receptor subcellular redistribution and expression of major metabolic genes. Blood triglycerides and fatty acid composition were also measured to assess hepatic Δ9 desaturase activity. The results showed that fructose diet increased blood triglycerides and Δ9 desaturase activity. On the other hand, stress led to corticosterone elevation, glucocorticoid receptor activation and decrease in adipocyte size, while phosphoenolpyruvate carboxykinase, adipose tissue triglyceride lipase, FAT/CD36 and sterol regulatory element binding protein-1c (SREBP-1c) were increased, pointing to VAT lipolysis and glyceroneogenesis. The combination of stress and fructose diet was associated with marked stimulation of fatty acid synthase and acetyl-CoA carboxylase mRNA level and with increased 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase protein levels, suggesting a coordinated increase in hexose monophosphate shunt and de novo lipogenesis. It however did not influence the level of peroxisome proliferator-activated receptor-gamma, SREBP-1c and carbohydrate responsive element-binding protein. In conclusion, our results showed that only combination of dietary fructose and stress increase glucocorticoid prereceptor metabolism and stimulates lipogenic enzyme expression suggesting that interaction between stress and fructose may be instrumental in promoting VAT expansion and dysfunction. Copyright © 2018 Elsevier B.V. All rights reserved.

Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium

NARCIS (Netherlands)

Biagiotti, E.; Bosch, K. S.; Ninfali, P.; Frederiks, W. M.; van Noorden, C. J.

2000-01-01

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown, it may be related;either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized

Effect of Xylitol on Growth of Streptococcus pneumoniae in the Presence of Fructose and Sorbitol

Science.gov (United States)

Tapiainen, Terhi; Kontiokari, Tero; Sammalkivi, Laura; Ikäheimo, Irma; Koskela, Markku; Uhari, Matti

2001-01-01

Xylitol is effective in preventing acute otitis media by inhibiting the growth of Streptococcus pneumoniae. To clarify this inhibition we used fructose, which is known to block similar growth inhibition observed in Streptococcus mutans. In addition, we evaluated the efficacy of sorbitol in inhibiting the growth of pneumococci, as sorbitol is widely used for indications similar to those for which xylitol is used. The addition of 5% xylitol to the growth medium resulted in marked growth inhibition, an effect which was totally eliminated in the presence of 1, 2.5, or 5% fructose but not in the presence of 1 or 5% glucose, 1% galactose, or 1% sucrose. This finding implies that xylitol-induced inhibition of pneumococcal growth is mediated via the fructose phosphotransferase system in a way similar to that in which mutans group streptococcal growth is inhibited. The addition of sorbitol at concentrations of 1, 2.5, or 5% to the growth medium did not affect the growth of pneumococci and neither inhibited nor enhanced the xylitol-induced growth impairment. Thus, it seems that xylitol is the only commercially used sugar substitute proven to have an antimicrobial effect on pneumococci. PMID:11120960

Glucose-6-phosphate dehydrogenase activity decreases during storage of leukoreduced red blood cells

NARCIS (Netherlands)

Peters, Anna L.; van Bruggen, Robin; de Korte, Dirk; van Noorden, Cornelis J. F.; Vlaar, Alexander P. J.

2016-01-01

During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity

Determination of the hydrothermal degradation products of D-(U-14C) glucose and D-(U-14C) fructose by TLC

International Nuclear Information System (INIS)

Bonn, G.; Bobleter, O.

1983-01-01

Hydrothermal degradation was examined using D-(U- 14 C) glucose and D-(U- 14 C) fructose. By thin layer chromatography with methylene chloride, tetrahydrofuran (THF), acetic acid - 60:20:20 as a mobile phase; it was possible to separate and identify the carbohydrates and their reaction products, glyceraldehyde, dihydroxyacetone, methylglyoxal, glycolaldehyde, 5-hydroxymethylfurfural and furfural. Up to 99% of the initial activity was determined by scintillation counting of the TL-chromatograms. A reaction scheme for the hydrothermal degradation of glucose and fructose was obtained from these results. (author)

Structures of Staphylococcus aureus D-tagatose-6-phosphate kinase implicate domain motions in specificity and mechanism.

Science.gov (United States)

Miallau, Linda; Hunter, William N; McSweeney, Sean M; Leonard, Gordon A

2007-07-06

High resolution structures of Staphylococcus aureus d-tagatose-6-phosphate kinase (LacC) in two crystal forms are herein reported. The structures define LacC in apoform, in binary complexes with ADP or the co-factor analogue AMP-PNP, and in a ternary complex with AMP-PNP and D-tagatose-6-phosphate. The tertiary structure of the LacC monomer, which is closely related to other members of the pfkB subfamily of carbohydrate kinases, is composed of a large alpha/beta core domain and a smaller, largely beta "lid." Four extended polypeptide segments connect these two domains. Dimerization of LacC occurs via interactions between lid domains, which come together to form a beta-clasp structure. Residues from both subunits contribute to substrate binding. LacC adopts a closed structure required for phosphoryl transfer only when both substrate and co-factor are bound. A reaction mechanism similar to that used by other phosphoryl transferases is proposed, although unusually, when both substrate and co-factor are bound to the enzyme two Mg(2+) ions are observed in the active site. A new motif of amino acid sequence conservation common to the pfkB subfamily of carbohydrate kinases is identified.

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways.

Science.gov (United States)

Zeng, Lin; Chakraborty, Brinta; Farivar, Tanaz; Burne, Robert A

2017-11-01

The glucose/mannose-phosphotransferase system (PTS) permease EII Man encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EII Fru , respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EII Man Expression of genes for EII Man and EII Fru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN Carbohydrate transport by EII Man had a negative influence on expression of manLMN but not fruRKI In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression. IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and

An in Situ NMR Study of the Mechanism for the Catalytic Conversion of Fructose to 5-Hydroxymethylfurfural and then to Levulinic Acid Using ¹³ C Labeled d -Fructose

Energy Technology Data Exchange (ETDEWEB)

Zhang, Jing [Department of Chemistry and Institute for Atom Efficient; Weitz, Eric [Department of Chemistry and Institute for Atom Efficient

2012-04-26

The pathways for the formation of 5-hydroxymethylfurfural (HMF) by dehydration of d-fructose and for the formation of levulinic acid and formic acid from HMF by rehydration were investigated by in situ13C and 1H NMR using both unlabeled and 13C-labeled fructose. Water or DMSO was used as the solvent with Amberlyst 70, PO43–/niobic acid, or sulfuric acid as catalysts. Only HMF is observed using NMR for fructose dehydration in DMSO with any of the three catalysts or without a catalyst. For each system, results with 13C-labeled fructose indicate that the first carbon (C-1) or sixth carbon (C-6) of fructose maps onto the corresponding carbons of HMF. For fructose dehydration in H2O with a PO43–/niobic acid catalyst, in addition to HMF, furfural was observed as a product. However, we show that furfural is not a reaction product deriving from HMF under our conditions. Rather our data indicate that there is a parallel reaction pathway open to fructose when the reaction takes place in H2O with a PO43–/niobic acid catalyst. The corresponding 13C-labeled results show that the first carbon in fructose maps onto the first carbon (aldehyde carbon) in furfural. Using 13C-enriched HMF formed from dehydration of 13C-labeled fructose in DMSO or H2O, we investigated the pathway for HMF rehydration to levulinic and formic acid. The data in different solvents and with different catalysts are consistent with a common mechanism for HMF rehydration, which results in the C-1 and C-6 carbon of HMF being transformed to the carbon of formic acid and methyl carbon (C-5) of levulinic acid, respectively.

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

Science.gov (United States)

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Inhibition of Anaerobic Phosphate Release by Nitric Oxide in Activated Sludge

Science.gov (United States)

Van Niel, E. W. J.; Appeldoorn, K. J.; Zehnder, A. J. B.; Kortstee, G. J. J.

1998-01-01

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P · g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-β-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used. PMID:9687452

Apparent heat capacity measurements and thermodynamic functions of D(−)-fructose by standard and temperature-modulated calorimetry

International Nuclear Information System (INIS)

Magoń, A.; Pyda, M.

2013-01-01

Highlights: ► Experimental, apparent heat capacity of fructose was investigated by advanced thermal analysis. ► Equilibrium melting parameters of fructose were determined. ► Decomposition, superheating of crystalline fructose during melting process were presented. ► TGA, DSC, and TMDSC are useful tools for characterisation of fructose. - Abstract: The qualitative and quantitative thermal analyses of crystalline and amorphous D(−)-fructose were studied utilising methods of standard differential scanning calorimetry (DSC), quasi-isothermal temperature-modulated differential scanning calorimetry (quasi-isothermal TMDSC), and thermogravimetric analysis (TGA). Advanced thermal analysis of fructose was performed based on heat capacity. The apparent total and apparent reversing heat capacities, as well as phase transition parameters were examined on heating and cooling. The melting temperature, T m , of crystalline D(−)-fructose shows a heating rate dependency, which increases with raising the heating rate and leads to superheating. The equilibrium melting temperatures: T m ∘ (onset) = 370 K and T m ∘ (peak) = 372 K, and the equilibrium enthalpy of fusion Δ fus H ° = 30.30 kJ · mol −1 , of crystalline D(−)-fructose were estimated on heating for the results at zero heating rate. Anomalies in the heat capacity in the liquid state of D(−)-fructose, assigned as possible tautomerisation equilibrium, were analysed by DSC and quasi-isothermal TMDSC, both on heating and cooling. Thermal stability of crystals in the region of the melting temperature was examined by TGA and quasi-isothermal TMDSC. Melting, mutarotation, and degradation processes occur simultaneously and there are differences in values of the liquid heat capacity of D(−)-fructose with varied thermal history, measured by quasi-isothermal TMDSC. Annealing of amorphous D(−)-fructose between the glass transition temperature, T g , and the melting temperature, T m , also leads to

High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation

Energy Technology Data Exchange (ETDEWEB)

Tan, Xiaoying [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Xu, Xingbo [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Zeisberg, Elisabeth M. [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany); Zeisberg, Michael, E-mail: mzeisberg@med.uni-goettingen.de [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany)

2016-04-08

Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones. - Highlights: • We exposed human kidney fibroblasts to media containing 1 mM or 3 mM phosphate. • Increased phosphate influx causes phosphorylation of DNA methyltransferase Dnmt1. • Phosphorylated Dnmt1 causes promoter methylation and transcriptional silencing of RASAL1. • Depletion of RASAL1 causes increased intrinsic Ras-GTP activity and fibroblast activation. • Inorganic phosphate causes fibroblast activation independent of phospho-regulatory hormones.

Leptin and glucocorticoid signaling pathways in the hypothalamus of female and male fructose-fed rats

Directory of Open Access Journals (Sweden)

Vojnović-Milutinović Danijela

2014-01-01

Full Text Available Alterations in leptin and glucocorticoid signaling pathways in the hypothalamus of male and female rats subjected to a fructose-enriched diet were studied. The level of expression of the key components of the leptin signaling pathway (neuropeptide Y /NPY/ and suppressor of cytokine signaling 3 /SOCS3/, and the glucocorticoid signaling pathway (glucocorticoid receptor /GR/, 11β-hydroxysteroid dehydrogenase type 1 /11βHSD1/ and hexose-6-phosphate dehydrogenase /H6PDH/ did not differ between fructose-fed rats and control animals of both genders. However, in females, a fructose-enriched diet provoked increases in the adiposity index, plasma leptin and triglyceride concentrations, and displayed a tendency to decrease the leptin receptor (ObRb protein and mRNA levels. In male rats, the fructose diet caused elevations in plasma non-esterified fatty acids and triglycerides, as well as in both plasma and hypothalamic leptin concentrations. Our results suggest that a fructose-enriched diet can induce hyperleptinemia in both female and male rats, but with a more pronounced effect on hypothalamic leptin sensitivity in females, probably contributing to the observed development of visceral adiposity. [Projekat Ministarstva nauke Republike Srbije, br. III41009

The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood

Science.gov (United States)

Valdes, Kayla M.; Sundar, Ganesh S.; Vega, Luis A.; Belew, Ashton T.; Islam, Emrul; Binet, Rachel; El-Sayed, Najib M.

2016-01-01

Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the fru locus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fru operon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-d-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment. PMID:26787724

Biochemical characterization of a D-psicose 3-epimerase from Treponema primitia ZAS-1 and its application on enzymatic production of D-psicose.

Science.gov (United States)

Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2016-01-15

The rare sugar D-psicose is a hexoketose monosaccharide and a C-3 epimer of D-fructose. D-Psicose is a novel functional sweetener with 70% of the sweetness but only 0.3% of the energy content of sucrose. Generally, the industrial production of D-psicose involves a bioconversion from D-fructose induced by ketose 3-epimerases. The D-psicose 3-epimerase (DPEase) gene from Treponema primitia ZAS-1 (Trpr-DPEase) was cloned and overexpressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified with a molecular mass of 33 kDa. Trpr-DPEase exhibited optimal activity at pH 8.0 and 70 °C and was sensitive to temperature, with relative thermal stability below 50 °C. It was strictly metal-dependent and displayed maximum catalytic activity with 450 µmol L(-1) Co(2+). The Km values of the enzyme for D-psicose and D-fructose were 209 and 279 mmol L(-1) respectively. The D-psicose/D-fructose equilibrium ratio of Trpr-DPEase was 28:72. A novel DPEase from T. primitia ZAS-1 was characterized that could catalyze the formation of D-psicose from D-fructose. D-Psicose was produced at a yield of 137.5 g L(-1) from 500 g L(-1) D-fructose, suggesting that Trpr-DPEase might be appropriate for the industrial production of D-psicose. © 2015 Society of Chemical Industry.

Complex formation between uranium(VI) and α-D-glucose 1-phosphate

International Nuclear Information System (INIS)

Koban, A.; Geipel, G.; Bernhard, G.

2003-01-01

The complex formation of uranium(VI) with α-D-glucose 1-phosphate (C 6 H 11 O 6 PO 3 2- , G1P) was determined by time-resolved laser-induced fluorescence spectroscopy (TRLFS) at pH 4 and potentiometric titration in the pH range from 3 to 10. Both measurements show the formation of a 1 : 1 complex at lower pH values. The formation constant of UO 2 (C 6 H 11 O 6 PO 3 ) was calculated from TRLFS measurements to be log β 11 = 5.72±0.12, and from potentiometric titration log β 11 = 5.40±0.25, respectively. It was found by potentiometric titration that at higher pH values the complexation changes to a 1 : 2 complex. The stability constant for this complex was calculated to be log β 12 = 8.96±0.18. (orig.)

Enzymatic synthesis of rare sugars with L-rhamnulose-1-phosphate aldolase from Thermotoga maritima MSB8.

Science.gov (United States)

Li, Zijie; Wu, Xiaoru; Cai, Li; Duan, Shenglin; Liu, Jia; Yuan, Peng; Nakanishi, Hideki; Gao, Xiao-Dong

2015-09-15

L-Rhamnulose-1-phosphate aldolase from a thermophilic source (Thermotoga maritima MSB8) (RhaDT.mari) was heterologously overexpressed in Escherichia coli and the stereoselectivity of this enzyme with or without Nus tag was investigated. We also applied this enzyme to the synthesis of rare sugars D-psicose, D-sorbose, L-tagatose and L-fructose using our one-pot four-enzyme system. To the best of our knowledge, this is the first use of RhaD from a thermophilic source for rare sugar synthesis and the temperature tolerance of this enzyme paves the path for large scale fermentation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Felodipine attenuates vascular inflammation in a fructose-induced rat model of metabolic syndrome via the inhibition of NF-kappaB activation.

Science.gov (United States)

Tan, Hong-wei; Xing, Shan-shan; Bi, Xiu-ping; Li, Li; Gong, Hui-ping; Zhong, Ming; Zhang, Yun; Zhang, Wei

2008-09-01

Metabolic syndrome is associated with an increased incidence of atherosclerosis. Clinical studies have shown that calcium channel blockers (CCB) inhibit the progression of atherosclerosis. However, the underlying mechanism is unclear. We investigated the inhibitory effect of felodipine on adhesion molecular expression and macrophage infiltration in the aorta of high fructose-fed rats (FFR). Male Wistar rats were given 10% fructose in drinking water. After 32 weeks of high fructose feeding, they were treated with felodipine (5 mg x kg(-1) x d(-1)) for 6 weeks. The control rats were given a normal diet and water. The aortic expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the infiltration of macrophages were measured by real-time RT-PCR and/or immunohistochemistry. NF-kappaB activity was measured by electrophoretic mobility shift assay (EMSA). After 32 weeks of high fructose feeding, FFR displayed increased body weight, systolic blood pressure (SBP), serum insulin, and triglycerides when compared with the control rats. The aortic expressions of ICAM-1 and VCAM-1 were significantly increased in FFR than in the control rats and accompanied by the increased activity of NF-kappaB. FFR also showed significantly increased CD68- positive macrophages in the aortic wall. After treatment with felodipine, SBP, serum insulin, and the homeostasis model assessment decreased significantly. In addition to reducing ICAM-1 and VCAM-1, felodipine decreased macrophages in the aortic wall. EMSA revealed that felodipine inhibited NF-kappaB activation in FFR. Felodipine inhibited vessel wall inflammation. The inhibition of NF-kappaB may be involved in the modulation of vascular inflammatory response by CCB in metabolic syndrome.

Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.

Science.gov (United States)

Li, Zijie; Cai, Li; Qi, Qingsheng; Styslinger, Thomas J; Zhao, Guohui; Wang, Peng George

2011-09-01

We report herein a one-pot four-enzyme approach for the synthesis of the rare sugars d-psicose, d-sorbose, l-tagatose, and l-fructose with aldolase FucA from a thermophilic source (Thermus thermophilus HB8). Importantly, the cheap starting material DL-GP (DL-glycerol 3-phosphate), was used to significantly reduce the synthetic cost. Copyright © 2011 Elsevier Ltd. All rights reserved.

Oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary 1α-hydroxycholecalciferol.

Science.gov (United States)

Bobeck, Elizabeth A; Hellestad, Erica M; Helvig, Christian F; Petkovich, P Martin; Cook, Mark E

2016-03-01

While it is well established that active vitamin D treatment increases dietary phytate phosphate utilization, the mechanism by which intestinal alkaline phosphatase (IAP) participates in phytate phosphate use is less clear. The ability of human IAP (hIAP) oral antibodies to prevent dietary phytate phosphate utilization in the presence of 1α-hydroxycholecalciferol (1α-(OH) D3) in a chick model was investigated. hIAP specific chicken immunoglobulin Y (IgY) antibodies were generated by inoculating laying hens with 17 synthetic peptides derived from the human IAP amino acid sequence and harvesting egg yolk. Western blot analysis showed all antibodies recognized hIAP and 6 of the 8 antibodies selected showed modest inhibition of hIAP activity in vitro (6 to 33% inhibition). In chicks where dietary phosphate was primarily in the form of phytate, 4 selected hIAP antibodies inhibited 1α-(OH) D3-induced increases in blood phosphate, one of which, generated against selected peptide (MFPMGTPD), was as effective as sevelamer hydrochloride in preventing the 1α-(OH) D3-induced increase in blood phosphate, but ineffective in preventing an increase in body weight gain and bone ash induced by 1α-(OH) D3. These studies demonstrated that orally-delivered antibodies to IAP limit dietary phytate-phosphate utilization in chicks treated with 1α-(OH) D3, and implicate IAP as an important host enzyme in increasing phytate phosphate bioavailability in 1α-(OH) D3 fed chicks. © 2015 Poultry Science Association Inc.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum

DEFF Research Database (Denmark)

Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh

2016-01-01

is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose...... uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate...... kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed...

Evaluation of the In Vivo and In Vitro Effects of Fructose on Respiratory Chain Complexes in Tissues of Young Rats

Directory of Open Access Journals (Sweden)

Ernesto António Macongonde

2015-01-01

Full Text Available Hereditary fructose intolerance (HFI is an autosomal-recessive disorder characterized by fructose and fructose-1-phosphate accumulation in tissues and biological fluids of patients. This disease results from a deficiency of aldolase B, which metabolizes fructose in the liver, kidney, and small intestine. We here investigated the effect of acute fructose administration on the activities of mitochondrial respiratory chain complexes, succinate dehydrogenase (SDH, and malate dehydrogenase (MDH in cerebral cortex, liver, kidney, and skeletal muscle of male 30-day-old Wistar rats. The rats received subcutaneous injection of sodium chloride (0.9%; control group or fructose solution (5 μmol/g; treated group. One hour later, the animals were euthanized and the cerebral cortex, liver, kidney, and skeletal muscle were isolated and homogenized for the investigations. Acute fructose administration increased complex I-III activity in liver. On the other hand, decreased complexes II and II-III activities in skeletal muscle and MDH in kidney were found. Interestingly, none of these parameters were affected in vitro. Our present data indicate that fructose administration elicits impairment of mitochondrial energy metabolism, which may contribute to the pathogenesis of the HFI patients.

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase - Investigation into an Apparent Loss of Stereospecificity

Energy Technology Data Exchange (ETDEWEB)

LowKam, C.; Liotard, B; Sygusch, J

2010-01-01

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a ({alpha}/{beta}){sub 8} fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys{sup 205}, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.

[Kinetic properties of the fructose influx across the brush border of the rat jejunum. Effects of a diet rich in fructose].

Science.gov (United States)

Crouzoulon, G

1978-10-01

The unidirectional influx (i.e. initial rate of uptake) of D-fructose across the brush border of rat jejunum is a saturable function of concentration, with a Kt of 125 mM, which implicates a carrier mechanism. This mechanism appears to be very specific for fructose in view of the lack of influx inhibition observed in the presence of large concentrations of the sugars or polyols, D-glucose, D-galactose, D-mannose, D-xylose, L-sorbose, D-tagatose, sorbitol or mannitol. D-Fructose uptake is inhibited by incubation, preceded by a 30-min preincubation in the same inhibitory conditions, in the absence of Na, or in the presence of metabolic poisons, NaF, 2,4-dinitrophenol, monoiodoacetate. Phloridzin (10-3 M), with or without preincubation, has no effect on uptake. D-Fructose influx is stimulated by fructose feeding, mainly because the augmentation of the number of active sites of transfer: Jmax is increased two-fold, Kt is more weakly affected.

Thermodynamics of the hydrolysis reactions of α-D-galactose 1-phosphate, sn-glycerol 3-phosphate, 4-nitrophenyl phosphate, phosphocreatine, and 3-phospho-D-glycerate

International Nuclear Information System (INIS)

Goldberg, Robert N.; Lang, Brian E.; Lo, Catherine; Ross, David J.; Tewari, Yadu B.

2009-01-01

Microcalorimetry, high-performance liquid chromatography (h.p.l.c.), and an enzymatic assay have been used to conduct a thermodynamic investigation of five phosphate hydrolysis reactions: {α-D-galactose 1-phosphate(aq) + H 2 O(l) = D-galactose(aq) + orthophosphate(aq)} (1), {sn-glycerol 3-phosphate(aq) + H 2 O(l) = glycerol(aq) + orthophosphate(aq)} (2), {4-nitrophenyl phosphate(aq) + H 2 O(l) = 4-nitrophenol(aq) + orthophosphate(aq)} (3), {phosphocreatine(aq) + H 2 O(l) = creatine(aq) + orthophosphate(aq)} (4), and {3-phospho-D-glycerate(aq) + H 2 O(l) = D-glycerate(aq) + orthophosphate(aq)} (5). Calorimetrically determined enthalpies of reaction Δ r H(cal) were measured for reactions (1)-(5) and the apparent equilibrium constant K' was measured for reaction (2). The pKs and standard enthalpies of reaction Δ r H 0 for the H + and Mg 2+ binding reactions of the reactants and products in the aforementioned reactions were obtained either from the literature or by estimation. A chemical equilibrium model was then used to calculate standard equilibrium constants K and standard enthalpies of reaction Δ r H 0 for chemical reference reactions that correspond to the overall biochemical reactions that were studied experimentally. Property values from the literature and thermodynamic network calculations were used to obtain values of the equilibrium constants for the chemical reference reactions that correspond to the overall biochemical reactions (1). These values were compared with other results from the literature and also correlated with structural features. The results obtained in this study can be used in the chemical equilibrium model to calculate values of K', the standard apparent Gibbs free energy changes Δ r G '0 , the standard apparent enthalpy changes Δ r H '0 , changes in binding of the proton Δ r N(H + ), and the position of equilibrium for the overall biochemical reactions considered in this study over a reasonably wide range of temperature, pH, p

NFKB activity decreased in BALB/c mice with high fat diet and fructose

Science.gov (United States)

Nur'aini, Farida Dewi; Rahayu, Sri; Rifa'i, Muhaimin

2017-05-01

Excessive consumption of fat and fructose leads to obesity due to lipid accumulation. The excessive lipid causes hypertrophy in the adipocytes which lead to cell death. Consequently, dead adipocytes will produce adipokines, which cause macrophages and lymphocytes to infiltrate into the adipose tissue, elevating pro-inflammatory cytokines, thus triggering the production of pro-inflammatory cytokines through NFκB activity. Elicited soybeans extract (ESE) with bacteria and light contain Glyceollin and Isoflavones, which inhibit the activation of NFKB and reduce plasma cholesterol levels by upregulating cholesterol metabolism. This study aimed to analyze the effect of ESE against the relative number of CD4+ NFκB+ cells in BALB/c mice spleen after administrated by high-fat diet food and fructose (HFD) for 20 weeks. Mice were given orally with ESE after administrated by HFD at dose 78 mg/kgBW (D1), 104 mg/kgBW (D2), and 130 mg/kgBW (D3) for 4 weeks. This study also used positive control (HFD mice model without ESE treatment) and normal mice. Identification of NFKB activation was conducted using Flowcytometry analytical methods. Our result indicated that ESE could decrease significantly activation of NFκB in CD4 cell compare than positive control. The optimum dose that can decrease the relative number of CD4+ NFκB+ cells is dose 3.

SwissProt search result: AK107595 [KOME

Lifescience Database Archive (English)

Full Text Available AK107595 002-131-A11 (Q6FZH6) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_BARQU 2e-11 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q8DJI6) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_SYNEL 6e-49 ...

Phosphoenolpyruvate-Dependent Fructose Phosphotransferase System of Rhodopseudomonas sphaeroides : Purification and Physicochemical and Immunochemical Characterization of a Membrane-Associated Enzyme I

NARCIS (Netherlands)

Brouwer, Marius; Elferink, Marieke G.L.; Robillard, George T.

1982-01-01

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called “soluble factor” (SF). SF has been partially purified by a combination of hydrophobic interaction and

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

Science.gov (United States)

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q6HPL2) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_BACHK 3e-41 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q98LX5) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_RHILO 6e-90 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q9HT00) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_HALSA 6e-44 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (O86781) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_STRCO 2e-50 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (P57963) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_PASMU 3e-98 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q56275) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_THIFE 3e-82 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (P17169) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_ECOLI 4e-72 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (P40831) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_MYCLE 5e-62 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (O57981) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_PYRHO 3e-59 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (P40831) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_MYCLE 9e-47 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (O68956) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_MYCSM 2e-62 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q6F6U8) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_ACIAD 4e-88 ...

High fructose-mediated attenuation of insulin receptor signaling does not affect PDGF-induced proliferative signaling in vascular smooth muscle cells.

Science.gov (United States)

Osman, Islam; Poulose, Ninu; Ganapathy, Vadivel; Segar, Lakshman

2016-11-15

Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[ 14 C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5-25mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia. Copyright © 2016 Elsevier B.V. All rights reserved.

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q8DRA8) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_STRR6 4e-54 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (P72720) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_SYNY3 4e-49 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q56213) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_THET8 5e-55 ...

ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

Directory of Open Access Journals (Sweden)

Maria José Leandro

Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

A convenient photosynthesis of uniformly [14C]-labelled D-glucose, D-fructose and sucrose, and chemical synthesis of methyl-α-D-glucopyranoside ([U-14C]-glucose)

International Nuclear Information System (INIS)

Srinivas, G.; Unny, V.K.P.; Mukkanti, K.; Choudary, B.M.

2013-01-01

This paper describes a convenient procedure for the radiochemical preparation of D-[U- 14 C]-glucose, D-[U- 14 C]-fructose and [U- 14 C]-sucrose with high specific activity by photosynthesis using ‘canna indica’ leaf, [ 14 C]-carbon dioxide and water in presence of light in a closed system. The [ 14 C]-sugars formed were extracted, separated and then purified by paper chromatography. Further, the pure D-[U- 14 C]-glucose obtained was converted to methyl-α-D-glucopyranoside ([U- 14 C]-glucose) by glycosidation with methanol using (i) HCl, the conventional Fischer method (ii) heterogeneous organic cation exchange resin (Amberlite IR-120 (H + )) and (iii) heterogeneous inorganic cation exchanged montmorillonites called metal M +n -monts. The results indicated that the latter in the form of Fe +3 -montmorillonite gave a better yield ( 65%) as compared to others (40–56%). The radiochemical purity of the no-carrier added product was more than 98%. The product retained its specific activity as that of the starting material which is in the range of 250–300 mCi/mmole (9.25–11.1 GBq/mmole), suitable for use as a radiotracer in biochemical investigations. - Graphical abstract: A convenient photosynthesis of uniformly [ 14 C]-labelled D-glucose, D-fructose and sucrose, and chemical synthesis of methyl-α-D-glucopyranoside ([U- 14 C]-glucose)/[ 14 C]–AMG, in short. The photosynthesis of D-[U- 14 C]-glucose, and two other [ 14 C]-sugars (fructose and sucrose) with high specific activity using ‘Canna indica’ leaf, 14 CO 2 and water in presence of light is presented in this paper. Further, the D-[U- 14 C]-glucose obtained was converted to methyl-α-D-glucopyranoside ([U- 14 C]-glucose)/[ 14 C]–AMG having 98% radiochemical purity and specific activity in the range of 250–300 mCi/mmole, using montmorillonites (M +n -monts). Highlights: ► Synthesis of methyl α D-glucopyranoside ([U- 14 C] glucose) has not been reported using M +n -monts. ► M +n -monts are

Aqueous seed extract of Hunteria umbellata (K. Schum.) Hallier f. (Apocynaceae) palliates hyperglycemia, insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome in rats.

Science.gov (United States)

Ajiboye, T O; Hussaini, A A; Nafiu, B Y; Ibitoye, O B

2017-02-23

Hunteria umbellata is used in the management and treatment of diabetes and obesity in Nigeria. This study evaluates the effect of aqueous seed extract of Hunteria umbellata on insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome MATERIALS AND METHODS: Rats were randomized into seven groups (A-G). Control (group A) and group C rats received control diet for nine weeks while rats in groups B, D - G were placed on high-fructose diet for 9 weeks. In addition to the diets, groups C - F rats orally received 400, 100, 200 and 400mg/kg body weight aqueous seed extract of Hunteria umbellata for 3 weeks starting from 6th - 9th week. High-fructose diet (when compared to control rats) mediated a significant (phigh-density lipoprotein cholesterol was decreased significantly. Levels of proinflammatory factor, tumour necrosis factor-α, interleukin-6 and 8 were also increased by the high fructose diet. Moreover, it mediated decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and level of glutathione reduced. Conversely, levels of malondialdehyde, conjugated dienes, lipid hydroperoxides, protein carbonyl and fragmented DNA were elevated. Aqueous seed extract of Hunteria umbellata significantly ameliorated the high fructose diet-mediated alterations. From this study, it is concluded that aqueous seed extract of Hunteria umbellata possesses hypoglycemic, hypolipidemic and antioxidants abilities as evident from its capability to extenuate insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome rats. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q9Z6U0) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CHLPN 2e-51 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q9JWN9) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_NEIMA 3e-66 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q8CX33) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_SHEON 3e-68 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q9PH05) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_XYLFA 3e-64 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q8RG65) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_FUSNN 2e-47 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q8NND3) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CORGL 2e-90 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q7MP62) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_VIBVY 4e-69 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q7NIG8) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_GLOVI 3e-62 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q8XHZ7) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_CLOPE 9e-55 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q8CX33) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_SHEON 4e-53 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q8CY30) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_BRUSU 4e-69 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q5FUY5) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_GLUOX 4e-49 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q81VN5) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_BACAN 2e-41 ...

SwissProt search result: AK107318 [KOME

Lifescience Database Archive (English)

Full Text Available AK107318 002-126-E03 (Q8DEF3) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_VIBVU 9e-70 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q9KUM8) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_VIBCH 9e-52 ...

SwissProt search result: AK105856 [KOME

Lifescience Database Archive (English)

Full Text Available AK105856 001-203-H06 (Q5QZH5) Glucosamine--fructose-6-phosphate aminotransferase [i...) (L-glutamine-D-fructose-6-phosphate amidotransferase) (Glucosamine-6-ph GLMS_IDILO 2e-51 ...

Optimization of Liquid Medium for High Phosphate Solubilization by Serratia Marcescens Strain AGKT4

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Mohd Yusoff Abd. Samad

2017-12-01

Full Text Available This study is on the optimization of the medium for solubilization of phosphate based on the Box-Behnken design and response surface methodology. Optimization of the liquid medium for phosphate solubilization using Serratia marcescens strain AGKT4 was carried out by varying the concentrations of 3 ingredients; the fructose, peptone and inoculum size of bacteria. A mathematical model derived from the response surface methodology was then validated statistically for the target test variables. The highest phosphate solubilization in the medium was achieved at the optimal concentrations of fructose and peptone at 6% (w/v and 0.6% (w/v, respectively. The maximum phosphate solubilization at these concentrations was 239.12 µg/mL. Under the same conditions, the bacterial growth in the medium was 9 log10 CFU.

Fructose diet alleviates acetaminophen-induced hepatotoxicity in mice.

Science.gov (United States)

Cho, Sungjoon; Tripathi, Ashutosh; Chlipala, George; Green, Stefan; Lee, Hyunwoo; Chang, Eugene B; Jeong, Hyunyoung

2017-01-01

Acetaminophen (APAP) is a commonly used analgesic and antipyretic that can cause hepatotoxicity due to production of toxic metabolites via cytochrome P450 (Cyp) 1a2 and Cyp2e1. Previous studies have shown conflicting effects of fructose (the major component in Western diet) on the susceptibility to APAP-induced hepatotoxicity. To evaluate the role of fructose-supplemented diet in modulating the extent of APAP-induced liver injury, male C57BL/6J mice were given 30% (w/v) fructose in water (or regular water) for 8 weeks, followed by oral administration of APAP. APAP-induced liver injury (determined by serum levels of liver enzymes) was decreased by two-fold in mice pretreated with fructose. Fructose-treated mice exhibited (~1.5 fold) higher basal glutathione levels and (~2 fold) lower basal (mRNA and activity) levels of Cyp1a2 and Cyp2e1, suggesting decreased bioactivation of APAP and increased detoxification of toxic metabolite in fructose-fed mice. Hepatic mRNA expression of heat shock protein 70 was also found increased in fructose-fed mice. Analysis of bacterial 16S rRNA gene amplicons from the cecal samples of vehicle groups showed that the fructose diet altered gut bacterial community, leading to increased α-diversity. The abundance of several bacterial taxa including the genus Anaerostipes was found to be significantly correlated with the levels of hepatic Cyp2e1, Cyp1a2 mRNA, and glutathione. Together, these results suggest that the fructose-supplemented diet decreases APAP-induced liver injury in mice, in part by reducing metabolic activation of APAP and inducing detoxification of toxic metabolites, potentially through altered composition of gut microbiota.

Fructose diet alleviates acetaminophen-induced hepatotoxicity in mice.

Directory of Open Access Journals (Sweden)

Sungjoon Cho

Full Text Available Acetaminophen (APAP is a commonly used analgesic and antipyretic that can cause hepatotoxicity due to production of toxic metabolites via cytochrome P450 (Cyp 1a2 and Cyp2e1. Previous studies have shown conflicting effects of fructose (the major component in Western diet on the susceptibility to APAP-induced hepatotoxicity. To evaluate the role of fructose-supplemented diet in modulating the extent of APAP-induced liver injury, male C57BL/6J mice were given 30% (w/v fructose in water (or regular water for 8 weeks, followed by oral administration of APAP. APAP-induced liver injury (determined by serum levels of liver enzymes was decreased by two-fold in mice pretreated with fructose. Fructose-treated mice exhibited (~1.5 fold higher basal glutathione levels and (~2 fold lower basal (mRNA and activity levels of Cyp1a2 and Cyp2e1, suggesting decreased bioactivation of APAP and increased detoxification of toxic metabolite in fructose-fed mice. Hepatic mRNA expression of heat shock protein 70 was also found increased in fructose-fed mice. Analysis of bacterial 16S rRNA gene amplicons from the cecal samples of vehicle groups showed that the fructose diet altered gut bacterial community, leading to increased α-diversity. The abundance of several bacterial taxa including the genus Anaerostipes was found to be significantly correlated with the levels of hepatic Cyp2e1, Cyp1a2 mRNA, and glutathione. Together, these results suggest that the fructose-supplemented diet decreases APAP-induced liver injury in mice, in part by reducing metabolic activation of APAP and inducing detoxification of toxic metabolites, potentially through altered composition of gut microbiota.

Photosystem II excitation pressure and photosynthetic carbon metabolism in Chlorella vulgaris

International Nuclear Information System (INIS)

Savitch, L.V.; Maxwell, D.P.; Huner, N.P.A.

1996-01-01

Chlorella vulgaris grown at 5 degrees C/150 micromoles m -2 s -1 mimics cells grown under high irradiance (27 degrees C/2200 micromoles m -2 s -1 ). This has been rationalized through the suggestion that both populations of cells were exposed to comparable photosystem II (PSII) excitation pressures measured as the chlorophyll a fluorescence quenching parameter, 1 - qP (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687-694). To assess the possible role(s) of feedback mechanisms on PSII excitation pressure, stromal and cytosolic carbon metabolism were examined. Sucrose phosphate synthase and fructose-1,6-bisphosphatase activities as well as the ratios of fructose-1,6-bisphosphate/fructose-6 phosphate and sucrose/starch indicated that cells grown at 27 degrees C/2200 micromoles m -2 s -1 appeared to exhibit a restriction in starch metabolism. In contrast, cells grown at 5 degrees C/150 micromoles-1 m -2 s -1 appeared to exhibit a restriction in the sucrose metabolism based on decreased cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase activities as well as a low sucrose/starch ratio. These metabolic restrictions may feedback on photosynthetic electron transport and, thus, contribute to the observed PSII excitation pressure. We conclude that, although PSII excitation pressure may reflect redox regulation of photosynthetic acclimation to light and temperature in C. vulgaris, it cannot be considered the primary redox signal. Alternative metabolic sensing/signaling mechanisms are discussed

Dietary fructose and risk of metabolic syndrome in adults: Tehran Lipid and Glucose study.

Science.gov (United States)

Hosseini-Esfahani, Firoozeh; Bahadoran, Zahra; Mirmiran, Parvin; Hosseinpour-Niazi, Somayeh; Hosseinpanah, Farhad; Azizi, Fereidoun

2011-07-12

Studies have shown that the excessive fructose intake may induce adverse metabolic effects. There is no direct evidence from epidemiological studies to clarify the association between usual amounts of fructose intake and the metabolic syndrome. The aim this study was to determine the association of fructose intake and prevalence of metabolic syndrome (MetS) and its components in Tehranian adults. This cross-sectional population based study was conducted on 2537 subjects (45% men) aged 19-70 y, participants of the Tehran Lipid and Glucose Study (2006-2008). Dietary data were collected using a validated 168 item semi-quantitative food frequency questionnaire. Dietary fructose intake was calculated by sum of natural fructose (NF) in fruits and vegetables and added fructose (AF) in commercial foods. MetS was defined according to the modified NCEP ATP III for Iranian adults. The mean ages of men and women were 40.5 ± 13.6 and 38.6 ± 12.8 years, respectively. Mean total dietary fructose intakes were 46.5 ± 24.5 (NF: 19.6 ± 10.7 and AF: 26.9 ± 13.9) and 37.3 ± 24.2 g/d (NF: 18.6 ± 10.5 and AF: 18.7 ± 13.6) in men and women, respectively. Compared with those in the lowest quartile of fructose intakes, men and women in the highest quartile, respectively, had 33% (95% CI, 1.15-1.47) and 20% (95% CI, 1.09-1.27) higher risk of the metabolic syndrome; 39% (CI, 1.16-1.63) and 20% (CI, 1.07-1.27) higher risk of abdominal obesity; 11% (CI, 1.02-1.17) and 9% (CI, 1.02-1.14) higher risk of hypertension; and 9% (CI, 1-1.15) and 9% (1.04-1.12) higher risk of impaired fasting glucose. Higher consumption of dietary fructose may have adverse metabolic effects.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

Science.gov (United States)

Smith, Clyde A.; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B.

2014-01-01

The bifunctional acetyltransferase(6′)-Ie-phosphotransfer­ase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a ‘gatekeeper’ residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme. PMID:24914967

No differential effect of beverages sweetened with fructose, high-fructose corn syrup, or glucose on systemic or adipose tissue inflammation in normal-weight to obese adults: a randomized controlled trial1

Science.gov (United States)

Cromer, Gail; Breymeyer, Kara L; Roth, Christian L; Weigle, David S

2016-01-01

Background: Sugar-sweetened beverage (SSB) consumption and low-grade chronic inflammation are both independently associated with type 2 diabetes and cardiovascular disease. Fructose, a major component of SSBs, may acutely trigger inflammation, which may be one link between SSB consumption and cardiometabolic disease. Objective: We sought to determine whether beverages sweetened with fructose, high-fructose corn syrup (HFCS), and glucose differentially influence systemic inflammation [fasting plasma C-reactive protein and interleukin-6 (IL-6) as primary endpoints] acutely and before major changes in body weight. Secondary endpoints included adipose tissue inflammation, intestinal permeability, and plasma fetuin-A as potential mechanistic links between fructose intake and low-grade inflammation. Design: We conducted a randomized, controlled, double-blind, crossover design dietary intervention (the Diet and Systemic Inflammation Study) in 24 normal-weight to obese adults without fructose malabsorption. Participants drank 4 servings/d of fructose-, glucose-, or HFCS-sweetened beverages accounting for 25% of estimated calorie requirements while consuming a standardized diet ad libitum for three 8-d periods. Results: Subjects consumed 116% of their estimated calorie requirement while drinking the beverages with no difference in total energy intake or body weight between groups as reported previously. Fasting plasma concentrations of C-reactive protein and IL-6 did not differ significantly at the end of the 3 diet periods. We did not detect a consistent differential effect of the diets on measures of adipose tissue inflammation except for adiponectin gene expression in adipose tissue (P = 0.005), which was lowest after the glucose phase. We also did not detect consistent evidence of a differential impact of these sugars on measures of intestinal permeability (lactulose:mannitol test, plasma zonulin, and plasma lipopolysaccharide-binding protein). Conclusion: Excessive

Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

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Cliona M Stapleton

2011-04-01

Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

Structural basis for phosphatidylinositol-phosphate biosynthesis

Science.gov (United States)

Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

2015-10-01

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

Time course of radiolabeled 2-deoxy-D-glucose 6-phosphate turnover in cerebral cortex of goats

International Nuclear Information System (INIS)

Pelligrino, D.A.; Miletich, D.J.; Albrecht, R.F.

1987-01-01

The vivo dephosphorylation rate of 2-deoxy-D-glucose 6-phosphate (DGP) in the cerebral cortex of goats injected intravenously with radiolabeled 2-deoxy-D-glucose (DG) was investigated. Serial rapidly frozen samples of parietal cortical gray tissue were obtained at regular intervals over time periods from 45 min to 3 h in awake goats or in paralyzed and artificially ventilated goats maintained under 70% N 2 O or pentobarbital sodium anesthesia. The samples were analyzed for glucose content and separate DG and DGP activities. The rate parameters for phosphorylation (k/sup */ 4 ) and dephosphorylation (k/sup */ 4 ) were estimated in each animal. The glucose phosphorylation rate (PR) was calculated over the intervals 3-5 (or 6), 3-10, 3-20, 3-30, and 3-45 min, assuming k/sup */ 4 = O. As the evaluation period was extended beyond 10 min, the calculated PR became increasingly less when compared with that calculated over the 3- to 5- (or 6) min interval (PR/sub i/). Furthermore, as metabolic activity decreased, the magnitude of the error increased such that at 45 min pentobarbital-anesthetize goats underestimated the PR/sub i/ by 46.5% compared with only 23.1 in N 2 O-anesthetized goats. This was also reflected in the >twofold higher k/sup */ 4 /k/sup */ 3 ratio in the pentobarbital vs. N 2 O-anesthetized group. It is concluded that when using the DG method in the goat, DGP dephosphorylation cannot be ignored when employing >10-min evaluation periods

Disruption of the Candida albicans TPS1 Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity†

Science.gov (United States)

Zaragoza, Oscar; Blazquez, Miguel A.; Gancedo, Carlos

1998-01-01

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 106 CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation. PMID:9683476

Magnesium isoglycyrrhizinate blocks fructose-induced hepatic NF-κB/NLRP3 inflammasome activation and lipid metabolism disorder.

Science.gov (United States)

Zhao, Xiao-Juan; Yang, Yan-Zi; Zheng, Yan-Jing; Wang, Shan-Chun; Gu, Hong-Mei; Pan, Ying; Wang, Shui-Juan; Xu, Hong-Jiang; Kong, Ling-Dong

2017-08-15

Magnesium isoglycyrrhizinate as a hepatoprotective agent possesses immune modulation and anti-inflammation, and treats liver diseases. But its effects on immunological-inflammatory and metabolic profiles for metabolic syndrome with liver injury and underlying potential mechanisms are not fully understood. In this study, magnesium isoglycyrrhizinate alleviated liver inflammation and lipid accumulation in fructose-fed rats with metabolic syndrome. It also suppressed hepatic inflammatory signaling activation by reducing protein levels of phosphorylation of nuclear factor-kappa B p65 (p-NF-κB p65), inhibitor of nuclear factor kappa-B kinase α/β (p-IKKα/β) and inhibitor of NF-κB α (p-IκBα) as well as nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and Caspase-1 in rats, being consistent with its reduction of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 levels. Furthermore, magnesium isoglycyrrhizinate modulated lipid metabolism-related genes characterized by up-regulating peroxisome proliferator-activated receptor-α (PPAR-α) and carnitine palmitoyl transferase-1 (CPT-1), and down-regulating sensor for fatty acids to control-1 (SREBP-1) and stearoyl-CoA desaturase 1 (SCD-1) in the liver of fructose-fed rats, resulting in the reduction of triglyceride and total cholesterol levels. These effective actions were further confirmed in fructose-exposed BRL-3A and HepG2 cells. The molecular mechanisms underpinning these observations suggest that magnesium isoglycyrrhizinate may inhibit NF-κB/NLRP3 inflammasome activation to reduce immunological-inflammatory response, which in turn may prevent liver lipid metabolic disorder and accumulation under high fructose condition. Thus, blockade of NF-κB/NLRP3 inflammasome activation and lipid metabolism disorder by magnesium isoglycyrrhizinate may be the potential therapeutic approach for improving fructose-induced liver injury with

Stannous Fluoride Effects on Gene Expression of Streptococcus mutans and Actinomyces viscosus.

Science.gov (United States)

Shi, Y; Li, R; White, D J; Biesbrock, A R

2018-02-01

A genome-wide transcriptional analysis was performed to elucidate the bacterial cellular response of Streptococcus mutans and Actinomyces viscosus to NaF and SnF 2 . The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of SnF 2 were predetermined before microarray study. Gene expression profiling microarray experiments were carried out in the absence (control) and presence (experimental) of 10 ppm and 100 ppm Sn 2+ (in the form of SnF 2 ) and fluoride controls for 10-min exposures (4 biological replicates/treatment). These Sn 2+ levels and treatment time were chosen because they have been shown to slow bacterial growth of S. mutans (10 ppm) and A. viscosus (100 ppm) without affecting cell viability. All data generated by microarray experiments were analyzed with bioinformatics tools by applying the following criteria: 1) a q value should be ≤0.05, and 2) an absolute fold change in transcript level should be ≥1.5. Microarray results showed SnF 2 significantly inhibited several genes encoding enzymes of the galactose pathway upon a 10-min exposure versus a negative control: lacA and lacB (A and B subunits of the galactose-6-P isomerase), lacC (tagatose-6-P kinase), lacD (tagatose-1,6-bP adolase), galK (galactokinase), galT (galactose-1-phosphate uridylyltransferase), and galE (UDP-glucose 4-epimerase). A gene fruK encoding fructose-1-phosphate kinase in the fructose pathway was also significantly inhibited. Several genes encoding fructose/mannose-specific enzyme IIABC components in the phosphotransferase system (PTS) were also downregulated, as was ldh encoding lactate dehydrogenase, a key enzyme involved in lactic acid synthesis. SnF 2 downregulated the transcription of most key enzyme genes involved in the galactose pathway and also suppressed several key genes involved in the PTS, which transports sugars into the cell in the first step of glycolysis.

Studies of alkaline mediated phosphate migration in synthetic phosphoethanolamine l-glycero-d-manno-heptoside derivatives

International Nuclear Information System (INIS)

Stewart, A.; Martin, A.; Richards, J.C.; Bernlind, C.; Oscarson, S.; Schweda, E.K.H.

1998-01-01

Synthetic 2-, 3-, 4- and 6-monophosphate derivatives of methyl α-d-mannopyranosides, the 4-, 6- and 7-monophosphate derivatives of methyl l-glycero-α-d-manno-heptopyranosides and the corresponding phosphoethanolamine derivatives and a 6,7-cyclic phosphate analogue of methyl l-glycero-α-d-manno-heptopyranoside were used to study phosphate migration and hydrolysis when subjected to strong alkaline conditions (4 M KOH, 120 C, 18 h). The resulting products were analyzed by 1 H NMR spectroscopy and electrospray mass spectrometry. It was found that phosphate substituents were stable under these conditions and neither migration nor hydrolysis was observed except for the heptose 7-phosphate, which gave a substantial amount of phosphate hydrolysis. In phosphoethanolamine-substituted compounds migration to adjacent positions with concomitant loss of ethanolamine was found together with hydrolysis. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

Czech Academy of Sciences Publication Activity Database

Šindelář, Luděk; Šindelářová, Milada

2002-01-01

Roč. 215, - (2002), s. 862-869 ISSN 0032-0935 R&D Projects: GA ČR GA522/99/1264 Institutional research plan: CEZ:AV0Z5038910 Keywords : Glucose 6 phosphate dehydrogenase * Nicotiana (viral infection) * Plant viruses Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.960, year: 2002

Glycogen synthase activation by sugars in isolated hepatocytes.

Science.gov (United States)

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

HO-1 Upregulation Attenuates Adipocyte Dysfunction, Obesity, and Isoprostane Levels in Mice Fed High Fructose Diets

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Zeid Khitan

2014-01-01

Full Text Available Background. Fructose metabolism is an unregulated metabolic pathway and excessive fructose consumption is known to activate ROS. HO-1 is a potent antioxidant gene that plays a key role in decreasing ROS and isoprostanes. We examined whether the fructose-mediated increase in adipocyte dysfunction involves an increase in isoprostanes and that pharmacological induction of HO-1 would decrease both isoprostane levels and adipogenesis. Methods and Results. We examined the effect of fructose, on adipogenesis in human MSCs in the presence and absence of CoPP, an inducer of HO-1. Fructose increased adipogenesis and the number of large lipid droplets while decreasing the number of small lipid droplets (P<0.05. Levels of heme and isoprostane in fructose treated MSC-derived adipocytes were increased. CoPP reversed these effects and markedly increased HO-1 and the Wnt signaling pathway. The high fructose diet increased heme levels in adipose tissue and increased circulating isoprostane levels (P<0.05 versus control. Fructose diets decreased HO-1 and adiponectin levels in adipose tissue. Induction of HO-1 by CoPP decreased isoprostane synthesis (P<0.05 versus fructose. Conclusion. Fructose treatment resulted in increased isoprostane production and adipocyte dysfunction, which was reversed by the increased expression of HO-1.

Dephosphorylation of 2-deoxyglucose 6-phosphate and 2-deoxyglucose export from cultured astrocytes.

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Forsyth, R J; Bartlett, K; Eyre, J

1996-03-01

Neurotransmitter-stimulated mobilization of astrocyte glycogen has been proposed as a basis for local energy homeostasis in brain. However, uncertainty remains over the fate of astrocyte glycogen. Upon transfer of cultured astrocytes pre-loaded with [2-3H]2-deoxyglucose 6-phosphate at non-tracer concentrations to a glucose-free, 2-deoxyglucose-free medium, rapid dephosphorylation of a proportion of the intracellular 2-deoxyglucose 6-phosphate pool and export of 2-deoxyglucose to the extracellular fluid occurs. Astrocytes show very low, basal rates of gluconeogenesis from pyruvate (approx. 1 nmol mg protein-1 h-1). Astrocytes in vivo may be capable of physiologically significant glucose export from glucose-6-phosphate. The low gluconeogenic activity in astrocytes suggests that the most likely source of glucose-6-phosphate may be glycogen. These findings support the hypothesis that export, as glucose, to adjacent neurons may be one of the possible fate(s) of astrocytic glycogen. Such export of glycogen as glucose occurring in response to increases in neuronal activity could contribute to energy homeostasis on a paracrine scale within brain.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis.

Science.gov (United States)

Narasaki, Craig T; Mertens, Katja; Samuel, James E

2011-01-01

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is

Fructose 1,6-biphosphate aldolase in the sera of Simmental young bulls Connection with the fattening capacity, muscle quantity and protein content.

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Križanović, D; Karadjole, I

1993-01-12

The activity of enzyme aldolase (ALD) was determined in the sera of Simmental young bulls, with the purpose to see whether there exists a relationship between the enzyme activity and fattening capacity and whether the ALD could be an indicator of the muscle quantity and protein content. Early ALD activity was correlated with slaughter weight and ADG in the last fattening month. The regression analysis suggests that young bulls with higher serum ALD acitivity at the start of the experiment had more total muscles and less bone in the analysed rib part. On the basis of ALD activity in the first fattening month it was possible to estimate protein and fat content in the MLD. ZUSAMMENFASSUNG: Fructose-1,6-biphosphat Aldolase beim Serum Simmentaler Jungbullen. Beziehung zwischen Mastfähigkeit, Muskelmenge und Proteingehalt Aldolase (ALD) Enzymtätigkeit im Serum von Simmentaler Jungbullen wurden in Hinblick auf eine Beziehung mit Mastfähigkeit, Muskelmenge und Proteingehalt untersucht. Es zeigt sich eine Korrelation zwischen früher ALD Aktivität und Schlachtgewicht und Tageszuwachs im Endmastmonat. Regressionsanalyse ergab, daß Jungbullen mit höherer ALD Aktivität bei Mastbeginn mehr Muskelanteil im m. longissimus dorsi und weniger Knochen besitzen. ALD Aktivität im ersten Mastmonat erlaubt Schätzung des Protein- und Fettgehalts im m. long, dorsi. RESUMEN: Fructosa 1,6-bifosfato aldolasa en el suero de los terneros simentales. Relatión con sus capacidades de ser cebados, su cantidad de músculos y el contenido de proteínas Con el fin de establecer la relación entre la actividad de enzimas y las capacidades durante el proceso de la ceba, asf como para precisar si el ALD puede servir como indicador de la cantidad de músculos y el contenido de proteinas, intentaba determinarse la actividad de los enzimas de aldolasa (ALD) en el suero de los terneros simentales. A base de la actividad indicial del ALD, es posible estimar el peso final de matadero y la crecencia

Dietary fructose and risk of metabolic syndrome in adults: Tehran Lipid and Glucose study

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Hosseinpanah Farhad

2011-07-01

Full Text Available Abstract Background Studies have shown that the excessive fructose intake may induce adverse metabolic effects. There is no direct evidence from epidemiological studies to clarify the association between usual amounts of fructose intake and the metabolic syndrome. Objective The aim this study was to determine the association of fructose intake and prevalence of metabolic syndrome (MetS and its components in Tehranian adults. Methods This cross-sectional population based study was conducted on 2537 subjects (45% men aged 19-70 y, participants of the Tehran Lipid and Glucose Study (2006-2008. Dietary data were collected using a validated 168 item semi-quantitative food frequency questionnaire. Dietary fructose intake was calculated by sum of natural fructose (NF in fruits and vegetables and added fructose (AF in commercial foods. MetS was defined according to the modified NCEP ATP III for Iranian adults. Results The mean ages of men and women were 40.5 ± 13.6 and 38.6 ± 12.8 years, respectively. Mean total dietary fructose intakes were 46.5 ± 24.5 (NF: 19.6 ± 10.7 and AF: 26.9 ± 13.9 and 37.3 ± 24.2 g/d (NF: 18.6 ± 10.5 and AF: 18.7 ± 13.6 in men and women, respectively. Compared with those in the lowest quartile of fructose intakes, men and women in the highest quartile, respectively, had 33% (95% CI, 1.15-1.47 and 20% (95% CI, 1.09-1.27 higher risk of the metabolic syndrome; 39% (CI, 1.16-1.63 and 20% (CI, 1.07-1.27 higher risk of abdominal obesity; 11% (CI, 1.02-1.17 and 9% (CI, 1.02-1.14 higher risk of hypertension; and 9% (CI, 1-1.15 and 9% (1.04-1.12 higher risk of impaired fasting glucose. Conclusion Higher consumption of dietary fructose may have adverse metabolic effects.

D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

International Nuclear Information System (INIS)

Akana, J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

2006-01-01

The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (β/α) 8 -barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn 2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn 2+ and inactive apoenzyme cannot be prepared, the affinity for Zn 2+ is decreased by alanine substitutions for the two histidine residues that coordinate the Zn 2+ ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn 2+ . The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn 2+ that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn 2+ and participate as acid/base catalysts are not conserved. We conclude that only the phosphate

D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

Energy Technology Data Exchange (ETDEWEB)

Akana,J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

2006-01-01

The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common ({beta}/{alpha}){sub 8}-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn{sup 2+} which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn{sup 2+} and inactive apoenzyme cannot be prepared, the affinity for Zn{sup 2+} is decreased by alanine substitutions for the two histidine residues that coordinate the Zn{sup 2+} ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn{sup 2+}. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn{sup 2+} that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn{sup 2+} and participate as acid/base catalysts

Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

Energy Technology Data Exchange (ETDEWEB)

Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

2009-05-14

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

Dual effects of fructose on ChREBP and FoxO1/3α are responsible for AldoB up-regulation and vascular remodelling.

Science.gov (United States)

Cao, Wei; Chang, Tuanjie; Li, Xiao-Qiang; Wang, Rui; Wu, Lingyun

2017-02-01

Increased production of methylglyoxal (MG) in vascular tissues is one of the causative factors for vascular remodelling in different subtypes of metabolic syndrome, including hypertension and insulin resistance. Fructose-induced up-regulation of aldolase B (AldoB) contributes to increased vascular MG production but the underlying mechanisms are unclear. Serum levels of MG and fructose were determined in diabetic patients with hypertension. MG level had significant positive correlations with blood pressure and fructose level respectively. C57BL/6 mice were fed with control or fructose-enriched diet for 3 months and ultrasonographic and histologic analyses were performed to evaluate arterial structural changes. Fructose-fed mice exhibited hypertension and high levels of serum MG with normal glucose level. Fructose intake increased blood vessel wall thickness and vascular smooth muscle cell (VSMC) proliferation. Western blotting and real-time PCR analysis revealed that AldoB level was significantly increased in both the aorta of fructose-fed mice and the fructose-treated VSMCs, whereas aldolase A (AldoA) expression was not changed. The knockdown of AldoB expression prevented fructose-induced MG overproduction and VSMC proliferation. Moreover, fructose significantly increased carbohydrate-responsive element-binding protein (ChREBP), phosphorylated FoxO1/3α and Akt1 levels. Fructose induced translocation of ChREBP from the cytosol to nucleus and activated AldoB gene expression, which was inhibited by the knockdown of ChREBP. Meanwhile, fructose caused FoxO1/3α shuttling from the nucleus to cytosol and inhibited its binding to AldoB promoter region. Fructose-induced AldoB up-regulation was suppressed by Akt1 inhibitor but enhanced by FoxO1/3α siRNA. Collectively, fructose activates ChREBP and inactivates FoxO1/3α pathways to up-regulate AldoB expression and MG production, leading to vascular remodelling. © 2017 The Author(s). published by Portland Press Limited on

Structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin

International Nuclear Information System (INIS)

Yajima, Shunsuke; Hara, Kodai; Iino, Daisuke; Sasaki, Yasuyuki; Kuzuyama, Tomohisa; Ohsawa, Kanju; Seto, Haruo

2007-01-01

The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg 2+ , NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR. The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg 2+ , NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg 2+ was shifted by NADPH in the active site

Arabidopsis CDS blastp result: AK105066 [KOME

Lifescience Database Archive (English)

Full Text Available AK105066 001-044-F12 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bisph...osphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 1e-166 ...

Fructose content in popular beverages made with and without high-fructose corn syrup.

Science.gov (United States)

Walker, Ryan W; Dumke, Kelly A; Goran, Michael I

2014-01-01

Excess fructose consumption is hypothesized to be associated with risk for metabolic disease. Actual fructose consumption levels are difficult to estimate because of the unlabeled quantity of fructose in beverages. The aims of this study were threefold: 1) re-examine the fructose content in previously tested beverages using two additional assay methods capable of detecting other sugars, especially maltose, 2) compare data across all methods to determine the actual free fructose-to-glucose ratio in beverages made either with or without high-fructose corn syrup (HFCS), and 3) expand the analysis to determine fructose content in commonly consumed juice products. Sugar-sweetened beverages (SSBs) and fruit juice drinks that were either made with or without HFCS were analyzed in separate, independent laboratories via three different methods to determine sugar profiles. For SSBs, the three independent laboratory methods showed consistent and reproducible results. In SSBs made with HFCS, fructose constituted 60.6% ± 2.7% of sugar content. In juices sweetened with HFCS, fructose accounted for 52.1% ± 5.9% of sugar content, although in some juices made from 100% fruit, fructose concentration reached 65.35 g/L accounting for 67% of sugars. Our results provide evidence of higher than expected amounts of free fructose in some beverages. Popular beverages made with HFCS have a fructose-to-glucose ratio of approximately 60:40, and thus contain 50% more fructose than glucose. Some pure fruit juices have twice as much fructose as glucose. These findings suggest that beverages made with HFCS and some juices have a sugar profile very different than sucrose, in which amounts of fructose and glucose are equivalent. Current dietary analyses may underestimate actual fructose consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

Science.gov (United States)

Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2002-05-01

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

International Nuclear Information System (INIS)

Blank, M.L.; Lee, T.; Cress, E.A.; Malone, B.; Fitzgerald, V.; Snyder, F.

1984-01-01

The metabolic pathway for 1-alkyl-2-acetyl-sn-glycerols, a recently discovered biologically active neutral lipid class, was elucidated in experiments conducted with rabbit platelets. The total lipid extract obtained from platelets incubated with 1-[1-,2- 3 H]alkyl-2-acetyl-sn-glycerols or 1-alkyl-2-[ 3 H]acetyl-sn-glycerols contained at least six metabolic products. The six metabolites, identified on the basis of chemical and enzymatic reactions combined with thin-layer or high-performance liquid chromatographic analyses, corresponded to 1-alkyl-sn-glycerols, 1-alkyl-2-acetyl-sn-glycero-3-phosphates, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholines, and 1-alkyl-2-actyl-sn-glycero-3-phosphocholines (platelet activating factor). These results indicate that the metabolic pathway for alkylacetylglycerols involves reaction steps catalyzed by the following enzymatic activities: choline- and ethanolamine- phosphotransferases, acetyl-hydrolase, an acyltransferase, and a phosphotransferase. The step responsible for the biosynthesis of platelet activating factor would appear to be the most important reaction in this pathway and this product could explain the hypotensive activities previously described for alkylacetyl-(or propionyl)-glycerols. Of particular interest was the preference exhibited for the utilization of the 1-hexadecyl-2-acetyl-sn-glycerol species in the formation of platelet activating factor

Genetics Home Reference: glucose-6-phosphate dehydrogenase deficiency

Science.gov (United States)

... deficiency Encyclopedia: Glucose-6-phosphate dehydrogenase test Encyclopedia: Hemolytic anemia Encyclopedia: Newborn jaundice Health Topic: Anemia Health Topic: G6PD Deficiency Health Topic: Newborn Screening Genetic and Rare Diseases Information Center (1 link) Glucose-6-phosphate dehydrogenase ...

Arabidopsis CDS blastp result: AK105724 [KOME

Lifescience Database Archive (English)

Full Text Available AK105724 001-201-G07 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bisph...osphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

Arabidopsis CDS blastp result: AK072243 [KOME

Lifescience Database Archive (English)

Full Text Available AK072243 J023003N10 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bispho...sphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

Arabidopsis CDS blastp result: AK065086 [KOME

Lifescience Database Archive (English)

Full Text Available AK065086 J013001L18 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bispho...sphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

Arabidopsis CDS blastp result: AK069285 [KOME

Lifescience Database Archive (English)

Full Text Available AK069285 J023011N22 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bispho...sphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

Purification and characterization of the d-xylose isomerase gene from Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Ho, N W.Y.; Rosenfeld, S; Stevis, P; Tsao, G T

1983-11-01

A DNA fragment containing both the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene and the D-xylulokinase (ATP: D-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The D-xylose isomerase gene was separated from the D-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The D-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the D-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first D-xylose isomerase gene that has been isolated and extensively purified from any organism. D-Xylose isomerase, the enzyme product of the D-xylose isomerase gene, is responsible for the conversion of D-xylose to D-xylulose, as well as D-glucose to D-fructose. It is widely believed that yeast cannot ferment D-xylose to ethanol primarily because of the lack of D-xylose isomerase in yeast. D-Xylose isomerase (also known as D-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the D-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting D-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology. 14 references.

Procedure and means for determining aminoglucoside phosphotransferase activity. Verfahren und Mittel zur Bestimmung der Aminoglucosid-Phosphotransferaseaktivitaet

Energy Technology Data Exchange (ETDEWEB)

Hunger, H D; Behrendt, G; Schmidt, G; Merkel, D

1988-02-10

A method and means are described for determining the activity of aminoglucoside phosphotransferase. Possible applications are genetic engineering, biotechnology and medicine. Cell lysates or cell extracts are contacted with a surface carrier on cellulose basis, in the presence of a mixture of ATP and /sup 32/P-ATP, with the phosphorylated kanamycin made visible in the solid phase by autoradiography.

Reactive Oxygen Species-Induced TXNIP Drives Fructose-Mediated Hepatic Inflammation and Lipid Accumulation Through NLRP3 Inflammasome Activation

Science.gov (United States)

Zhang, Xian; Zhang, Jian-Hua; Chen, Xu-Yang; Hu, Qing-Hua; Wang, Ming-Xing; Jin, Rui; Zhang, Qing-Yu; Wang, Wei; Wang, Rong; Kang, Lin-Lin; Li, Jin-Sheng; Li, Meng

2015-01-01

Abstract Aims: Increased fructose consumption predisposes the liver to nonalcoholic fatty liver disease (NAFLD), but the mechanisms are elusive. Thioredoxin-interacting protein (TXNIP) links oxidative stress to NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and this signaling axis may be involved in fructose-induced NAFLD. Here, we explore the role of reactive oxygen species (ROS)-induced TXNIP overexpression in fructose-mediated hepatic NLRP3 inflammasome activation, inflammation, and lipid accumulation. Results: Rats were fed a 10% fructose diet for 8 weeks and treated with allopurinol and quercetin during the last 4 weeks. Five millimolars of fructose-exposed hepatocytes (primary rat hepatocytes, rat hepatic parenchymal cells [RHPCs], HLO2, HepG2) were co-incubated with antioxidants or caspase-1 inhibitor or subjected to TXNIP or NLRP3 siRNA interference. Fructose induced NLRP3 inflammasome activation and pro-inflammatory cytokine secretion, janus-activated kinase 2/signal transducers and activators of transcription 3-mediated inflammatory signaling, and expression alteration of lipid metabolism-related genes in cultured hepatocytes and rat livers. NLRP3 silencing and caspase-1 suppression blocked these effects in primary rat hepatocytes and RHPCs, confirming that inflammasome activation alters hepatocyte lipid metabolism. Hepatocellular ROS and TXNIP were increased in animal and cell models. TXNIP silencing blocked NLRP3 inflammasome activation, inflammation, and lipid metabolism perturbations but not ROS induction in fructose-exposed hepatocytes, whereas antioxidants addition abrogated TXNIP induction and diminished the detrimental effects in fructose-exposed hepatocytes and rat livers. Innovation and Conclusions: This study provides a novel mechanism for fructose-induced NAFLD pathogenesis by which the ROS-TXNIP pathway mediates hepatocellular NLRP3 inflammasome activation, inflammation and lipid accumulation. Antioxidant

No difference in ad libitum energy intake in healthy men and women consuming beverages sweetened with fructose, glucose, or high-fructose corn syrup: a randomized trial1

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Kuzma, Jessica N; Cromer, Gail; Hagman, Derek K; Breymeyer, Kara L; Roth, Christian L; Foster-Schubert, Karen E; Holte, Sarah E; Callahan, Holly S; Weigle, David S; Kratz, Mario

2015-01-01

Background: Increased energy intake is consistently observed in individuals consuming sugar-sweetened beverages (SSBs), likely mainly because of an inadequate satiety response to liquid calories. However, SSBs have a high content of fructose, the consumption of which acutely fails to trigger responses in key signals involved in energy homeostasis. It is unclear whether the fructose content of SSBs contributes to the increased energy intake in individuals drinking SSBs. Objective: We investigated whether the relative amounts of fructose and glucose in SSBs modifies ad libitum energy intake over 8 d in healthy adults without fructose malabsorption. Design: We conducted 2 randomized, controlled, double-blind crossover studies to compare the effects of consuming 4 servings/d of a fructose-, glucose-, or aspartame-sweetened beverage (study A; n = 9) or a fructose-, glucose-, or high-fructose corn syrup (HFCS)–sweetened beverage (study B; n = 24) for 8 d on overall energy intake. SSBs were provided at 25% of estimated energy requirement, or an equivalent volume of the aspartame-sweetened beverage, and consumption was mandatory. All solid foods were provided at 125% of estimated energy requirements and were consumed ad libitum. Results: In study A, ad libitum energy intake was 120% ± 10%, 117% ± 12%, and 102% ± 15% of estimated energy requirements when subjects consumed the fructose-, glucose-, and aspartame-sweetened beverages. Energy intake was significantly higher in the fructose and glucose phases than in the aspartame phase (P fructose and glucose phases (P = 0.462). In study B, total energy intake during the fructose, HFCS, and glucose phases was 116% ± 14%, 116% ± 16%, and 116% ± 16% of the subject’s estimated total energy requirements (P = 0.880). Conclusions: In healthy adults, total 8-d ad libitum energy intake was increased in individuals consuming SSBs compared with aspartame-sweetened beverages. The energy overconsumption observed in individuals

Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii

International Nuclear Information System (INIS)

Arnfors, Linda; Hansen, Thomas; Meining, Winfried; Schönheit, Peter; Ladenstein, Rudolf

2005-01-01

Nucleoside kinase from the hyperthermophilic archaeon M. jannaschii is a member of the PFK-B family which belongs to the ribokinase superfamily. Here, its expression, purification, crystallization and preliminary X-ray analysis are described. Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg 2+ . The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 Å for the apoenzyme crystals and 1.93 Å for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer

Physiological role of glucose-6-phosphate dehydrogenase in cold acclimation of strawberry (Fragaria × ananassa)

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Zhang, Yong; Yu, Dingqun; Luo, Ya; Wang, Xiaorong; Chen, Qing; Sun, Bo; Wang, Yan; Liu, Zejing; Tang, Haoru

2018-04-01

In recent years, there has been an increasing interest in study of new resistance mechanism in fruit trees. All these regard the climate change and subsequent fruit production. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway (OPPP), and the expression of this enzyme is related to different biotic and abiotic stresses. Under accumulation of low temperature stress, the significant increase in G6PDH activity was found to be closely correlated to the levels of antioxidant enzymes, malondialdehyde (MDA) contents, sugar contents as well as changes of superoxide (O2•-). It is suggested that the enhancement of cold resistance of strawberry, which induced by cold acclimation, related to the significant increase in G6PDH activity. On one hand, G6PDH activates NADPH oxidase to generate reactive oxygen species (ROS); on the other hand, it may be involved in the activation of antioxidant enzymes, and accelerates many other important NADPH-dependent enzymatic reactions. Then further result in the elevation of membrane stability and cold resistance of strawberry. Interestingly, even though the plants were placed again under a temperature of 25°C for 1 d, the higher cold resistance, enzyme activities and soluble sugar content acquired.

Trehalose-6-phosphate synthesis controls yeast gluconeogenesis downstream and independent of SNF1.

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Deroover, Sofie; Ghillebert, Ruben; Broeckx, Tom; Winderickx, Joris; Rolland, Filip

2016-06-01

Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Δ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Δ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Δ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Δ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Arabidopsis CDS blastp result: AK106106 [KOME

Lifescience Database Archive (English)

Full Text Available AK106106 001-207-C12 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bisph...osphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 9e-39 ...

Self-assembled block copolymer photonic crystal for selective fructose detection.

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Ayyub, Omar B; Ibrahim, Michael B; Briber, Robert M; Kofinas, Peter

2013-08-15

The use of one-dimensional photonic crystals fabricated from a self-assembled lamellar block copolymer as a sensitive and selective fructose sensor is investigated. The polystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) films are functionalized with 2-(bromomethyl)phenylboronic acid. The boronic acid moiety confined within the lamellar morphology can reversibly bind to sugars such as fructose, imparting the photonic properties of the PS-b-P2VP film. The films exhibit a detection limit of 500 μM in water and 1mM in phosphate buffered saline. Exposure to a 50 mM solution of fructose invokes a highly visible color change from blue to orange. The films are also able to selectively recognize and respond to fructose in competitive studies in the presence of glucose, mannose and sucrose. Copyright © 2013 Elsevier B.V. All rights reserved.

Hydrogels based on polysaccharide-calcium phosphate with antibacterial / antitumor activity for 3D printing

Science.gov (United States)

Teterina, A. Yu; Fedotov, A. Yu; Zobkov, Yu V.; Sergeeva, N. S.; Sviridova, I. K.; Kirsanova, V. A.; Karalkin, P. A.; Komlev, V. S.

2018-04-01

The purpose of this study was to develop hydrogels for 3D printing of sodium alginate/gelatin/octacalcium phosphate-based constructs with antibacterial and antitumor activity intended for bone defects replacement in patients with malignant diseases. In this work, we evaluated the drug release kinetic and physico-chemical characteristics of constructs, as well as their specific activity, biocompatibility and osteoplastic properties by means of in vitro and in vivo tests. The principal possibility of creating the biocompatible bone substitutes with antibacterial/antitumor activity and osteoconductive-retaining properties of 3D printing method was demonstrated.

Preparation of D-[U-14C]galactose and α-D-[U-14C]galactose-1-phosphate

International Nuclear Information System (INIS)

Kolina, J.; Hromadkova, B.

1989-01-01

Optically pure D-[U- 14 C]galactose was prepared on a preparatory scale using the galactokinase enzyme. The suggested procedure allows to also prepare a α-D-[U- 14 C]galactose-1-phosphate and L-[U- 14 ]galactose giving good yield. The experiments proved that the raw fraction isolated from yeast of the Kluyveromyces fragilis strain or the Kluyveromyces lactis strain shows sufficient activity. Phosphorylation of D-[U- 14 C]galactose practically terminates after 30 mins of incubation. DL-[U- 14 C]galactose isolated using preparatory paper chromatography from the acid hydrolyzate of [U- 14 C] polysaccharide is a satisfactory radioactive precursor. The developed preparation procedure theoretically contributed towards the further elucidation of the problem of the proportional representation of galactose stereo-isomers in extracellular polysaccharide isolated from red algae. In this respect data in the literature differ and some sources state a significantly higher propertion of L-galactose. The experiments showed that [U- 14 C] polysaccharide isolated from the red algae Porphyridium cruentum prevalently contains D-[U- 14 C]galactose, which confirms the process of enzyme reaction. (author). 1 tab., 4 refs

Fructose downregulates miR-330 to induce renal inflammatory response and insulin signaling impairment: Attenuation by morin.

Science.gov (United States)

Gu, Ting-Ting; Song, Lin; Chen, Tian-Yu; Wang, Xing; Zhao, Xiao-Juan; Ding, Xiao-Qin; Yang, Yan-Zi; Pan, Ying; Zhang, Dong-Mei; Kong, Ling-Dong

2017-08-01

Fructose induces insulin resistance with kidney inflammation and injury. MicroRNAs are emerged as key regulators of insulin signaling. Morin has insulin-mimetic effect with the improvement of insulin resistance and kidney injury. This study investigated the protective mechanisms of morin against fructose-induced kidney injury, with particular focus on miR-330 expression change, inflammatory response, and insulin signaling impairment. miR-330, sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptor (S1PR)1/3 signaling, nuclear factor-κB (NF-κB)/NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, and insulin signaling were detected in kidney cortex of fructose-fed rats and fructose-exposed HK-2 cells, respectively. Whether miR-330 mediated inflammatory response to affect insulin signaling was examined using SphK1 inhibitor, S1PR1/3 short interfering RNA, or miR-330 mimic/inhibitor, respectively. Fructose was found to downregulate miR-330 expression to increase SphK1/S1P/S1PR1/3 signaling, and then activate NF-κB/NLRP3 inflammasome to produce IL-1β, causing insulin signaling impairment. Moreover, morin upregulated miR-330 and partly attenuated inflammatory response and insulin signaling impairment to alleviate kidney injury. These findings suggest that morin protects against fructose-induced kidney insulin signaling impairment by upregulating miR-330 to reduce inflammatory response. Morin may be a potential therapeutic agent for the treatment of kidney injury associated with fructose-induced inflammation and insulin signaling impairment. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.

Science.gov (United States)

Markmann, Sandra; Krambeck, Svenja; Hughes, Christopher J; Mirzaian, Mina; Aerts, Johannes M F G; Saftig, Paul; Schweizer, Michaela; Vissers, Johannes P C; Braulke, Thomas; Damme, Markus

2017-03-01

The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

International Nuclear Information System (INIS)

Martin, S.A.; Russell, J.B.

1987-01-01

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments, indicated that separate phosphotransferases systems existed for glucose, maltose, and sucrose. [ 14 C]maltose transport was inhibited by excess glucose and to a lesser extent by sucrose. [ 14 C]glucose and [ 14 C]sucrose transports were not inhibited by an excess of maltose. Since [ 14 C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of P/sub i/ was increased from 0 to 100 mM, a maltose phosphorylase was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for α-glucose 1-phosphate. Only sucrose-grown cells possessed sucrose hydrolase activity, and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities

Novel hydroxyamides and amides containing D-glucopyranose or D-fructose units: Biological assays in MCF-7 and MDST8 cell lines.

Science.gov (United States)

Carreiro, Elisabete P; Costa, Ana R; Cordeiro, Maria M; Martins, Rute; Pires, Tiago O; Saraiva, Mafalda; Antunes, Célia M; Burke, Anthony J

2016-02-01

A novel library of 15 compounds, hydroxyamides and amides containing a β-D-glucopyranose (D-Gluc) or a β-D-fructose (D-Fruc) units was designed and synthesized for antiproliferative assays in breast (MCF-7) and colon (MDST8) cancer cell lines. Twelve of them were hydroxyamides and were successfully synthesized from β-D-glucuronic acid (D-GluA). Six of these hydroxyamides which were acetylated hydroxy-β-D-glucopyranuronamide 2a-2f (1st Family) and the other six were their respective isomers, that is, hydroxy-β-D-fructuronamide 3a-3f (2nd Family), obtained by acid-base catalyzed isomerization. These compounds have the general structure, D-Gluc-C=ONH-CHR-(CH2)n-OH and D-Fruc-C=ONH-CHR-(CH2)n-OH, where R=an aromatic, alkyl or a hydrogen substituent, with n=0 or 1. Eight of these contained a chiral aminoalcohol group. Three compounds were amides containing a D-glucopyranose unit (3rd Family). SAR studies were conducted with these compounds. Antiproliferative studies showed that compound 4a, the bromo-amide containing the β-D-glucopyranose ring, potently inhibits the proliferation of the MDST8 cells. Five compounds (2e, 2f, 3d, 3e, and 3f) were shown to potently selectively inhibit the proliferation of the MCF-7 cells. Compound 4b was the only one showing inhibition in both cell lines. In general, the more active compounds were the amides and hydroxyamides containing the β-D-fructose moiety, and containing an alkyl group or hydrogen. Half-inhibitory concentrations (IC50) of between 0.01 and 10 μM, were observed. Copyright © 2015 Elsevier Ltd. All rights reserved.

(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.

Science.gov (United States)

Gebril, Hoda M; Avula, Bharathi; Wang, Yan-Hong; Khan, Ikhlas A; Jekabsons, Mika B

2016-02-01

Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a

Characterization of vascular complications in experimental model of fructose-induced metabolic syndrome.

Science.gov (United States)

El-Bassossy, Hany M; Dsokey, Nora; Fahmy, Ahmed

2014-12-01

Vascular dysfunction is an important complication associated with metabolic syndrome (MS). Here we fully characterized vascular complications in a rat model of fructose-induced MS. MS was induced by adding fructose (10%) to drinking water to male Wistar rats of 6 weeks age. Blood pressure (BP) and isolated aorta responses phenylephrine (PE), KCl, acetylcholine (ACh), and sodium nitroprusside (SNP) were recorded after 6, 9, and 12 weeks of fructose administration. In addition, serum levels of glucose, insulin, uric acid, tumor necrosis factor α (TNFα), lipids, advanced glycation end products (AGEs), and arginase activity were determined. Furthermore, aortic reactive oxygen species (ROS) generation, hemeoxygenase-1 expression, and collagen deposition were examined. Fructose administration resulted in a significant hyperinslinemia after 6 weeks which continued for 12 weeks. It was also associated with a significant increase in BP after 6 weeks which was stable for 12 weeks. Aorta isolated from MS animals showed exaggerated contractility to PE and KCl and impaired relaxation to ACh compared with control after 6 weeks which were clearer at 12 weeks of fructose administration. In addition, MS animals showed significant increases in serum levels of lipids, uric acid, AGEs, TNFα, and arginase enzyme activity after 12 weeks of fructose administration. Furthermore, aortae isolated from MS animals were characterized by increased ROS generation and collagen deposition. In conclusion, adding fructose (10%) to drinking water produces a model of MS with vascular complications after 12 weeks that are characterized by insulin resistance, hypertension, disturbed vascular reactivity and structure, hyperuricemia, dyslipidemia, and low-grade inflammation.

Influence of sulphate on effects of ADP, 3′,5′-cyclic amp and citrate on human platelet phosphofructokinase activity

NARCIS (Netherlands)

Akkerman, Jan Willem N.; Gorter, G.; Sixma, J.J.; Staal, Gerard E.J.

1974-01-01

1. 1. Sulphate ions activate partially-purified human platelet phosphofructokinase. This is caused by suppression of the cooperativity of the enzyme with respect to fructose 6-phosphate. 2. 2. Sulphate therefore markedly affects the influences of allosteric modifiers such as ADP, 3′,5′-cyclic AMP

Chemical and Enzymatic Approaches to Carbohydrate-Derived Spiroketals: Di-D-Fructose Dianhydrides (DFAs

Directory of Open Access Journals (Sweden)

José M. García Fernández

2008-08-01

Full Text Available Di-D-fructose dianhydrides (DFAs comprise a unique family of stereoisomeric spiro-tricyclic disaccharides formed upon thermal and/or acidic activation of sucroseand/ or D-fructose-rich materials. The recent discovery of the presence of DFAs in food products and their remarkable nutritional features has attracted considerable interest from the food industry. DFAs behave as low-caloric sweeteners and have proven to exert beneficial prebiotic nutritional functions, favouring the growth of Bifidobacterium spp. In the era of functional foods, investigation of the beneficial properties of DFAs has become an important issue. However, the complexity of the DFA mixtures formed during caramelization or roasting of carbohydrates by traditional procedures (up to 14 diastereomeric spiroketal cores makes evaluation of their individual properties a difficult challenge. Great effort has gone into the development of efficient procedures to obtain DFAs in pure form at laboratory and industrial scale. This paper is devoted to review the recent advances in the stereoselective synthesis of DFAs by means of chemical and enzymatic approaches, their scope, limitations, and complementarities.

Opposite lipemic response of Wistar rats and C57BL/6 mice to dietary glucose or fructose supplementation

Directory of Open Access Journals (Sweden)

C.R. Barbosa

2007-03-01

Full Text Available The metabolic effects of carbohydrate supplementation in mice have not been extensively studied. In rats, glucose- and fructose-rich diets induce hypertriacylglycerolemia. In the present study, we compared the metabolic responses to two monosaccharide supplementations in two murine models. Adult male Wistar rats (N = 80 and C57BL/6 mice (N = 60, after 3 weeks on a standardized diet, were submitted to dietary supplementation by gavage with glucose (G or fructose (F solutions (500 g/L, 8 g/kg body weight for 21 days. Glycemia was significantly higher in rats after fructose treatment (F: 7.9 vs 9.3 mM and in mice (G: 6.5 vs 10 and F: 6.6 vs 8.9 mM after both carbohydrate treatments. Triacylglycerolemia increased significantly 1.5 times in rats after G or F supplementation. Total cholesterol did not change with G treatment in rats, but did decrease after F supplementation (1.5 vs 1.4 mM, P < 0.05. Both supplementations in rats induced insulin resistance, as suggested by the higher Homeostasis Model Assessment Index. In contrast, mice showed significant decreases in triacylglycerol (G: 1.8 vs 1.4 and F: 1.9 vs 1.4 mM, P < 0.01 and total cholesterol levels (G and F: 2.7 vs 2.5 mM, P < 0.05 after both monosaccharide supplementations. Wistar rats and C57BL/6 mice, although belonging to the same family (Muridae, presented opposite responses to glucose and fructose supplementation regarding serum triacylglycerol, free fatty acids, and insulin levels after monosaccharide treatment. Thus, while Wistar rats developed features of plurimetabolic syndrome, C57BL/6 mice presented changes in serum biochemical profile considered to be healthier for the cardiovascular system.