Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

Os autores padronizaram métodos para a avaliação da atividade da glicose-6-fosfato desidrogenase e glutationa redutase. O princípio geral do primeiro método baseou-se na formação de metahemoglobina pelo nitrito de sódio, seguido da estimulação da via das pentoses pelo azul de metileno. Foram estudados 46 indivíduos adultos, sendo 23 do sexo masculino e 23 do feminino, não deficientes em glicose-6-fosfato desidrogenase (G6PD), com idades variando entre 20 e 30 anos. Os resultados revelaram que a redução da metahemoglobina pelo azul de metileno para sangue total, foram de 154.50 e 139.90 mg/min (p<0.05) respectivamente para o sexo masculino e feminino. Para hemácias lavadas os valores foram de 221.10 e 207.85 mg/min (n.s.) respectivamente. Estas observações permitiram concluir que ao se empregar hemácias lavadas e 0.7 g% de concentração de nitrito de sódio, por um lado não houve diferença entre os sexos e por outro, abreviou o tempo de leitura da quantidade residual de metahemoglobina para 90 minutos. A avaliação da atividade da glutationa redutase foi feita baseado no fato de que a cistamina (agente tiol) liga-se aos grupos SH da hemoglobina formando complexos. Estes complexos são revertidos pela ação da glutationa redutase, ocorrendo conjuntamente nesta reação a redução da metahemoglobina. Foram estudados 32 indivíduos adultos, sendo 16 do sexo masculino e 16 do feminino, não deficientes em G6PD, com idades variando entre 20 e 30 anos. Os resultados revelaram valores de redução da metahemoglobina pela cistamina de 81.27 e 91.13 mg/min (p<0.01) respectivamente para o sexo masculino e feminino. Estas observações permitiram concluir que o emprego de hemácias lavadas e 0.1 molar de concentração de cistamina torna possível a leitura da quantidade residual de metahemoglobina aos 180 minutos de incubação. A atividade da glutationa redutase avaliada por meio da redução da metahemoglobina pela cistamina, foi estudada em 14 indivíduos do sexo feminino antes e após o tratamento com 10 mg por dia de riboflavina durante 8 dias. Os resultados foram de 73.69 e 94.26 mg/min (p<0.01) antes e após o tratamento. Estas observações permitiram concluir que a oferta de riboflavina, mesmo para indivíduos normais, aumenta a atividade da glutationa redutase. Foram ainda avaliados 3 indivíduos da raça negra e deficientes em G6PD, sendo 2 do sexo masculino e 1 do feminino. Houve ativação parcial da G6PD e glutationa redutase, sendo estas alterações mais intensas nos indivíduos do sexo masculino. Considerando-se a raça e as características laboratoriais observadas, foi possível sugerir que a deficiência em G6PD verificada é do tipo Africano, bem como, permitiu considerar os indivíduos do sexo feminino coin o sendo heterozigoto para esta deficiência. Por fim, a análise dos resultados em seu conjunto permitiu concluir que os métodos propostos se mostraram eficientes para avaliar a atividade da G6PD e glutationa redutase. Esta última é dependente da via das pentoses, geradora de NADPH e da riboflavina, vitamina precursora de FAD.

Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females) aged 17 to 58 years. Twenty one (53.84%) of the patients presented a slow acetylating phenotype and 18 (46.16%) a fast acetylating phenotype. Glucose-6-phosphate-dehydrogenase (G6PD) activity was decreased in 5(23.80%) slow acetylators and in 4 (22.22%) fast acetylators. Glutathione reductase activity was decreased in 14 (66.66%) slow acetylators and in 12(66.66%) fast acetylators. Serum levels of free and total sulfadoxin were higher in slow acetylator (p _ 0.02). Analysis of the results permitted us to conclude that serum sulfadoxin levels are related to the acetylator phenotype. Furthermore, sulfadoxin levels were always above 50 μg/ml, a value considered therapeutic. Glutathione reductase deficiency observed in 66% of patients may be related to the intestinal malabsorption of nutrients, among them riboflavin, a FAD precursor vitamin, in patients with paracoceidioidomycosis.

Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

Glucose metabolism in the erythrocytes of the buffalo (Bubalus bubalis)

(CO2)-C-14 production from [1-C-14] glucose, the rate of glycolysis measured by the value of lactate production and the activities of various enzymes were determined in buffalo erythrocytes. Buffalo red cell glycolytic metabolites were estimated and used for the calculation of the mass action ratios of reactions catalyzed by the glycolytic enzymes of Bubalus bubalis. A comparison of the values of the mass action ratios with the equilibrium constants of the various glycolytic reactions indicate that hexokinase, phosphofructokinase, phosphoglycerate kinase and pyruvate kinase reactions are displaced from equilibrium, suggesting a regulatory role for each of these enzymes in buffalo erythrocyte glycolysis. (C) 1997 Elsevier B.V.

Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilization in growth medium

Co-inoculation of the fungus Aspergillus niger and the bacterium Burkholderia cepacia was undertaken to understand the interaction between different species of phosphate-solubilizing microorganisms (PSM). PSM were inoculated in a single or mixed (A. nigerB.similar to cepacia) culture. During 9 similar to days of incubation, microbial biomass was enhanced, accompanied with increases in the levels of soluble phosphate and titratable acidity, as well as increased acid phosphatase activity. Production of acids and levels of phosphate solubilization were greater in the co-culture of A.similar to nigerB.similar to cepacia than in the single culture. The quantity of phosphate solubilized by the co-culture ranged from 40.51 +/- 0.60 to 1103.64 +/- 1.21 similar to mu g similar to PO4 3-similar to mL-1 and was 922% higher than single cultures. pH of the medium dropped from 7.0 to 3.0 in the A.similar to niger culture, 3.1 in the co-culture, and 4.2 in the B.similar to cepacia culture. on the third day of postinoculation, acid production by the co-culture (mean 5.40 +/- 0.31 similar to mg NaOH mL-1) was 1990% greater than single cultures. Glucose concentration decreased almost completely (9799% of the starting concentration) by the ninth day of the incubation. These results show remarkable synergism by the co-culture in comparison with single cultures in the solubility of CaHPO4 under in vitro conditions. This synergy between microorganisms can be used in poor available phosphate soils to enhance phosphate solubilization.

Soluble phosphate accumulation by Aspergillus niger from fluorapatite

In order to determine conditions that may provide greater solubilization of insouluble phosphate, the fungus Aspergillus niger was grown in a stationary culture containing modified citrate medium supplemented with 800 mg fluorapatite per litre. Solubilization of insouluble phosphate increased with fungal growth, reaching a maximum after 11 days of culture. Soluble phosphate levels were correlated with pH of the culture medium but not with titratable acidity values, probably due to the metabolic activity of the fungus resulting from consumption of sugar in the culture medium. Fructose, glucose, xylose, and sucrose were the carbohydrates that favoured fluorapatite solubilization the most when compared with galactose and maltose. Although increasing fructose concentrations in the culture medium favoured mycelial growth, increased total acidity and a fall in pH, soluble phosphate levels were reduced, probably owing to consumption by the rapidly growing fungus. Among the nitrogen sources tested, ammonium salts favoured the production of larger amounts of soluble phosphate than organic nitrogen (peptone or urea) or nitrate, corresponding to the lowest pH and highest titratable acidity values obtained. © 1988 Springer-Verlag.

Glucocorticoids in vivo induce both insulin hypersecretion and enhanced glucose sensitivity of stimulus-secretion coupling in isolated rat islets

Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca2 2+ signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca2 2+signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally,weexplored the status of Ca2 2+-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase Cα as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the β-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity. Copyright © 2010 by The Endocrine Society.

Use of different carbon sources in cultivation of baker's yeast for production of glycerol-3-phosphate dehydrogenase

The physiological state of yeast cells changes during culture growth as a consequence of environmental changes (nutrient limitations, pH and metabolic products). Cultures that grow exponentially are heterogeneous cell populations made up of cells regulated by different metabolic and/or genetic control systems. The strain of baker's yeast selected by plating commercial compressed yeast was used for the production of glycerol-3- phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPD) has been widely used in the enzyme assays with diverse compounds of industrial interest, such as glycerol or glycerol phosphate, as well as a number of important bioanalytical applications. Each cell state determines the level of key enzymes (genetic control), fluxes through metabolic pathways (metabolic control), cell morphology and size. The present study was carried out to determine the effects of environmental conditions and carbon source on GPD production from baker's yeast. Glucose, glycerol, galactose and ethanol were used as carbon sources. Glycerol and ethanol assimilations required agitation, which was dependent on the medium volume in the fermentation flask for the greatest accumulation of intracellular GPD. Enzyme synthesis was also affected by the initial pH of the medium and inoculum size. The fermentation time required for a high level of enzyme formation decreased with the inoculum size. The greatest amount of enzyme (0.45 U/ml) was obtained with an initial pH of 4.5 in the medium containing ethanol or glycerol. The final pH was maintained in YP-ethanol, but in the YP-glycerol the final pH increased to 6.9 during growth.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Evaluation of glucose biosensors based on Prussian Blue and lyophilised, crystalline and cross-linked glucose oxidases (CLEC (R))

Glucose biosensors based on lyophilised, crystalline and cross-linked glucose oxidase (GOx, CLEC(R)) and commercially available lyophilised GOx immobilised on top of glassy carbon electrodes modified with electrodeposited Prussian Blue are critically compared. Two procedures were carried out for preparing the biosensors: (1) deposition of one layer of adsorbed GOx dissolved in an aqueous solution followed by deposition of two layers of low molecular weight Nafion(R) dissolved in 90% ethanol, and (2) deposition of two layers of a mixture of GOx with Nafion dissolved in 90% ethanol. The performance of the biosensors was evaluated in terms of linear response range for hydrogen peroxide and glucose, detection limit, and susceptibility to some common interfering species (ascorbic acid, acetaminophen and uric acid). The operational stability of the biosensors was evaluated by applying a steady potential of -50 mV versus Ag/AgCl to the glucose biosensor and injecting standard solutions of hydrogen peroxide and glucose (50 muM and 1.0 mM, respectively, in phosphate buffer) for at least 5 h in a flow-injection system. Scanning electron microscopy was used for visualisation of the Prussian Blue redox catalyst and in the presence of the different GOx preparations on the electrode surface. (C) 2001 Elsevier B.V. B.V. All rights reserved.