Efficient glycerol transformation by resting Gluconobacter cells.

In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by-product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.

Comparison of the compatible solute pool of two slightly halophilic planctomycetes species, Gimesia maris and Rubinisphaera brasiliensis.

Gimesia maris and Rubinisphaera brasiliensis are slightly halophilic representatives of the deep-branching phylum Planctomycetes. For osmoadaptation both species accumulated α-glutamate, sucrose, ectoine and hydroxyectoine. A major role was found for ectoine, hydroxyectoine as well as sucrose under hyper-osmotic shock conditions. Nevertheless, the levels of sucrose were up-regulated by the increased salinity levels and also by low nitrogen availability. Additionally, G. maris accumulated glucosylglycerate (GG) as major solute specifically under low nitrogen levels, which prompted us to analyse the transcript abundance of two homologues genes known for the biosynthesis of GG, namely glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). By qPCR using a suitable reference gene selected in this study, the transcript abundance of the biosynthetic genes was quantified in G. maris cells under hyper-osmotic shock or under low nitrogen conditions. The gpgS gene was induced under nitrogen-limiting conditions suggesting that GG synthesis is regulated primarily at the transcription level. Moreover, the expression of a gene coding for a putative sucrose-phosphorylase (Spase) located upstream the gpgS and gpgP genes was up-regulated, predicting a metabolic role of Spase probably related to GG synthesis.

D-glyceric aciduria.

Inherited metabolic diseases are a heterogeneous group of diseases caused by a punctual defect in cell metabolism, resulting in the accumulation of toxic intermediate metabolites or in the lack of important biomolecules for adequate cell functioning. D-glyceric aciduria is an inherited disease caused by a deficiency of glycerate 2-kinase activity, whose pathophysiological mechanisms remain unknown. The main clinical and neurological symptoms seen in affected patients include progressive encephalopathy, hypotonia, psychomotor and mental retardation, microcephaly, seizures, speech delay, metabolic acidosis, and even death. In this review we shall discuss these clinical and biochemical findings, as well as diagnosis and treatment of affected patients in order to raise awareness about this condition.

Unraveling the activation mechanism of the potato tuber ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.

Domain swapping between a cyanobacterial and a plant subunit ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.

Complete 1H and 13C NMR assignment of digeneaside, a low-molecular-mass carbohydrate produced by red seaweeds.

Digeneaside (alpha-D-mannopyranosyl-(1-->2)-D-glycerate) was extracted from the red algae, Bostrychia binderii, and purified by adsorption and gel-filtration chromatography. HPLC and ESI-MS techniques were used to follow purification steps and characterize digeneaside. NMR spectroscopy experiments (1D 1H, 13C, DEPT and 2D HMQC, COSY and TOCSY) were used to fully assign the 1H and 13C spectra.

Expression, purification, crystallization and preliminary crystallographic analysis of Leishmania mexicana phosphoglycerate mutase.

Bacterially expressed 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) from Leishmania mexicana with a six-His tag fused at its C-terminus was expressed from plasmid pET28a after IPTG induction in Escherichia coli cells and gave a yield of 20 mg of highly purified iPGAM per litre of cell culture. Crystals of the protein complexed with 3-phosphoglycerate were obtained by the hanging-drop method of vapour diffusion with PEG 4000 as the precipitating agent in the presence of cobalt chloride and diffracted synchrotron radiation to beyond 1.90 A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 72.27, c = 129.68 A. A model of Bacillus stearothermophilus iPGAM (33% identity) was used to provide an initial molecular-replacement solution. X-ray data to 2.05 A for the structure of L. mexicana iPGAM complexed with 2-phosphoglycerate have also been collected.

Ultrasensitive behavior in the synthesis of storage polysaccharides in cyanobacteria.

The glycogen synthetic pathway operates ultrasensitively as a function of the ADPglucose pyrophosphorylase (ADPGlcPPase) allosteric effectors, 3-phosphoglycerate and Pi, in permeabilized cells of the cyanobacterium Anabaena PCC 7120. In vitro data previously showed that the ultrasensitive behavior of ADPGlcPPase depends upon cross-talk between the two allosteric effectors, the enzyme's response being additionally modulated by molecular crowding [D.F. Gómez Casatiet al. (2000) Biochem J 350:139-147]. In the present work we show, experimentally and with a mathematical model, that alpha-1,4-glucan synthesis is also ultrasensitive in cells due to the propagation of the switch-like behavior of ADPGlcPPase to the synthetic pathway. Amplifications of up to 20-fold in storage-polysaccharide synthesis can be achieved with a modest 6.7-fold increase in 3-phosphoglycerate in the presence of 5 mM Pi in contrast to the 30-fold necessary in its absence. This is the first time that this phenomenon has been reported to occur in the glycogen synthetic pathway of a photosynthetic prokaryote. The implications of the results for plant cell physiology during light-dark transitions are discussed.

Coherent and robust modulation of a metabolic network by cytoskeletal organization and dynamics.

In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence. The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway. Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively. The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network. Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e. a high degree of polymerization relieves the negative control. The stability of the model's steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP. The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism.

ADP-glucose pyrophosphorylase from wheat endosperm. Purification and characterization of an enzyme with novel regulatory properties.

ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) was purified and characterized from two wheat (Triticum aestivum L.) tissues: leaf and endosperm. The leaf enzyme, purified over 1,300-fold, was found to be a heterotetramer composed of subunits of 51 and 54 kDa and possessing regulatory properties typical of AGPases from photosynthetic tissues, being mainly regulated by 3-phosphoglycerate (activator; A0.5=0.01 mM) and orthophosphate (inhibitor; I0.5=0.2 mM). Conversely, the enzyme from wheat endosperm was insensitive to activation by 3-phosphoglycerate and other metabolites. It was, however, inhibited by orthophosphate (I0.5=0.7 mM), ADP (I0.5=3.2 mM) and fructose-1,6-bisphosphate (0.5 = 1.5 mM). All of these inhibitory actions were reversed by 3-phosphoglycerate and fructose-6-phosphate. The endosperm enzyme was found to be a heterotetramer composed of subunits of 52 and 53 kDa, which were recognized by antiserum raised to spinach leaf AGPase. The results suggest that wheat endosperm AGPase possesses distinctive regulatory properties that are relevant in vivo.

Ultrasensitive glycogen synthesis in Cyanobacteria.

Cyanobacter ADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3-phosphoglycerate, elicited by orthophosphate and polyethyleneglycol-induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15-19-fold with respect to 1% of the maximal system velocity. Only a 2.4-3.8-fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200-fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.

Efectos beneficiosos del glicerofosfato de magnesio como tratamiento de la litiasis oxalocálcica en pacientes hipercalciúricos / Beneficial effect the magnesium salt of phosphoglyceric how treatment the lithiasis calcium oxalate in patients hypercalciuria

Para ensayar una nueva alternativa terapéutica de la hipercalciuria con litiasis oxalocálcica recidivante, se utilizó la sal magnésica del ácido glicerofosfórico (GPMg) en 24 pacientes (12 varones y 12 mujeres) con 36 años de edad promedio (rango: 20-63). El tipo de la hipercalciuria fue absortiva (A) o tabular (T) (n= 14 y 10 respectivamente). La dosis de PGMg fue de 3g diarios divididos en dos tomas. Se efectuaron controles clínicos a los 1-3 meses (período I), a los 4-6 meses (período II), a los 7-12 meses (período III). a los 12-18 meses (período IV) con determinaciones de Ca, P, Mg, Na, K, NH4, sulfato creatinina, ácidos úricos, cítrico y oxálico, pH y volumen urinario. Se calculó la saturación urinaria en términos de energía libre para la cristalización (DG) de oxalato de calcio, brushita, estruvita, ácido úrico y urato de sodio por medio del programa de computación EQUIL-AT. En un subgrupo de 7 pacientes (5 A y 2 T) se determinaron además la parathormona (PTH) sérica y el AMP cíclico en suero y orina en condiciones basales y al mes de tratamiento con GPMg. La calciuria disminuyo significativamente en todos los períodos considerados de ambos grupos (A y T). La diuresis se incrementó progresivamente. Otros componentes urinarios no presentaron cambios significativos. La DG para el oxalato de calcio y la brushita cayeron significativamente en todos los períodos. No hubo modificación en la saturación urinaria de los restantes compuestos. No hubo evidencias de hiperparatiroidismo secundario en el subgrupo controlado con determinación de PTH sérica y AMPc nefrógeno. En 7 pacientes tratados durante dos años hubo sólo un episodio de recurrencia. La tolerancia del GPMg fue excelente. (AU)

Efectos beneficiosos del glicerofosfato de magnesio como tratamiento de la litiasis oxalocálcica en pacientes hipercalciúricos / Beneficial effect the magnesium salt of phosphoglyceric how treatment the lithiasis calcium oxalate in patients hypercalciuria

Para ensayar una nueva alternativa terapéutica de la hipercalciuria con litiasis oxalocálcica recidivante, se utilizó la sal magnésica del ácido glicerofosfórico (GPMg) en 24 pacientes (12 varones y 12 mujeres) con 36 años de edad promedio (rango: 20-63). El tipo de la hipercalciuria fue absortiva (A) o tabular (T) (n= 14 y 10 respectivamente). La dosis de PGMg fue de 3g diarios divididos en dos tomas. Se efectuaron controles clínicos a los 1-3 meses (período I), a los 4-6 meses (período II), a los 7-12 meses (período III). a los 12-18 meses (período IV) con determinaciones de Ca, P, Mg, Na, K, NH4, sulfato creatinina, ácidos úricos, cítrico y oxálico, pH y volumen urinario. Se calculó la saturación urinaria en términos de energía libre para la cristalización (DG) de oxalato de calcio, brushita, estruvita, ácido úrico y urato de sodio por medio del programa de computación EQUIL-AT. En un subgrupo de 7 pacientes (5 A y 2 T) se determinaron además la parathormona (PTH) sérica y el AMP cíclico en suero y orina en condiciones basales y al mes de tratamiento con GPMg. La calciuria disminuyo significativamente en todos los períodos considerados de ambos grupos (A y T). La diuresis se incrementó progresivamente. Otros componentes urinarios no presentaron cambios significativos. La DG para el oxalato de calcio y la brushita cayeron significativamente en todos los períodos. No hubo modificación en la saturación urinaria de los restantes compuestos. No hubo evidencias de hiperparatiroidismo secundario en el subgrupo controlado con determinación de PTH sérica y AMPc nefrógeno. En 7 pacientes tratados durante dos años hubo sólo un episodio de recurrencia. La tolerancia del GPMg fue excelente. (AU)

Efectos beneficiosos del glicerofosfato de magnesio como tratamiento de la litiasis oxalocálcica en pacientes hipercalciúricos / Beneficial effect the magnesium salt of phosphoglyceric how treatment the lithiasis calcium oxalate in patients hypercalciuria

Para ensayar una nueva alternativa terapéutica de la hipercalciuria con litiasis oxalocálcica recidivante, se utilizó la sal magnésica del ácido glicerofosfórico (GPMg) en 24 pacientes (12 varones y 12 mujeres) con 36 años de edad promedio (rango: 20-63). El tipo de la hipercalciuria fue absortiva (A) o tabular (T) (n= 14 y 10 respectivamente). La dosis de PGMg fue de 3g diarios divididos en dos tomas. Se efectuaron controles clínicos a los 1-3 meses (período I), a los 4-6 meses (período II), a los 7-12 meses (período III). a los 12-18 meses (período IV) con determinaciones de Ca, P, Mg, Na, K, NH4, sulfato creatinina, ácidos úricos, cítrico y oxálico, pH y volumen urinario. Se calculó la saturación urinaria en términos de energía libre para la cristalización (DG) de oxalato de calcio, brushita, estruvita, ácido úrico y urato de sodio por medio del programa de computación EQUIL-AT. En un subgrupo de 7 pacientes (5 A y 2 T) se determinaron además la parathormona (PTH) sérica y el AMP cíclico en suero y orina en condiciones basales y al mes de tratamiento con GPMg. La calciuria disminuyo significativamente en todos los períodos considerados de ambos grupos (A y T). La diuresis se incrementó progresivamente. Otros componentes urinarios no presentaron cambios significativos. La DG para el oxalato de calcio y la brushita cayeron significativamente en todos los períodos. No hubo modificación en la saturación urinaria de los restantes compuestos. No hubo evidencias de hiperparatiroidismo secundario en el subgrupo controlado con determinación de PTH sérica y AMPc nefrógeno. En 7 pacientes tratados durante dos años hubo sólo un episodio de recurrencia. La tolerancia del GPMg fue excelente.

El test de lisis con glicerol acidificado, un método de screening para esferocitosis hereditaria: evaluación y valor diagnóstico / The scidified glycerol lysis test, a screening method for inherited spherocytosis

Se evaluó un método de screening para esferocitosis hereditaria, el test de lisis con Glicerol Acidificado (AGTL). En la evaluación se utilizaron 75 muestras de sangre de individuos normales (recién nacidos y adultos) y 20 patológicas (Esferocitosis Hereditaria, Insuficiencia Renal Crónica, Trastorno Hematológico Maligno y Daño Hepático). A base de los resultados obtenidos, se concluye lo siguiente: la temperatura de lectura más adecuada es 37-C; las muestras pueden ser conservadas por 24 hrs. a 4§C; los valores de referencia normal para recién nacidos y adultos son >2.100 y >1.200 segundos, respectivamente y que el método posee una sensibilidad de 100%

Human red cell glycolysis in high altitude chronic hypoxia.

We have found that glycolysis in human red blood cells under the hypoxic conditions found at high altitudes is connected with changes in enzyme activities and levels of various metabolic intermediates. The sensitivity of the four kinases to hypoxia results in 1) glycolytic hyperactivity leading to a higher intracellular energy state, and 2) accumulation of 2-3 DPG, whose role in the adaptation of red blood cell respiration to high altitude has been shown by previous research. PEP, 3PG , and G6P appear to be the main regulating intermediates in glycolysis in this system. The reason for the very large increase in G1- 6DP is still not clear.