Person: Pozueta Romero, Javier
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Pozueta Romero
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Javier
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Instituto de Agrobiotecnología (IdAB)
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0000-0002-0335-9663
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2094
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Publication Open Access Glycogen phosphorylase, the product of the glgP Gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli(American Society for Microbiology, 2006) Alonso Casajús, Nora; Dauvillee, David; Viale Bailone, Alejandro M.; Muñoz Pérez, Francisco José; Baroja Fernández, Edurne; Morán Zorzano, María Teresa; Eydallin, Gustavo; Ball, Steven; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaTo understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (ΔglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in ΔglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by ΔglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.Publication Open Access Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevents starch biosynthesis(National Academy of Sciences, 2000) Rodríguez López, Milagros; Baroja Fernández, Edurne; Zandueta Criado, Aitor; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaA distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated Keq* (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25°C is 110, and its standard-state free-energy change value (DG*) is 22.9 kcalymol (1 kcal 5 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (Km 5 0.5 mM). Pi and phosphorylated compounds such as 3-phosphoglycerate, PPi, ATP, ADP, NADP1, and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-a-D-glucan 4-a- D-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly blocking the starch biosynthesis.Publication Open Access Most of ADP-glucose linked to starch biosynthesis occurs outside the chloroplast in source leaves(National Academy of Sciences, 2004) Baroja Fernández, Edurne; Muñoz Pérez, Francisco José; Zandueta Criado, Aitor; Morán Zorzano, María Teresa; Viale Bailone, Alejandro M.; Alonso Casajús, Nora; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako GobernuaSucrose and starch are end products of two segregated gluconeogenic pathways, and their production takes place in the cytosol and chloroplast of green leaves, respectively. According to this view, the plastidial ADP glucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule ADPG. However, a growing body of evidences indicates that starch formation involves the import of cytosolic ADPG to the chloroplast. This evidence is consistent with the idea that synthesis of the ADPG linked to starch biosynthesis takes place in the cytosol by means of sucrose synthase, whereas AGP channels the glucose units derived from the starch breakdown. To test this hypothesis, we first investigated the subcellular localization of ADPG. Toward this end, we constructed transgenic potato plants that expressed the ADPG-cleaving adenosine diphosphate sugar pyrophosphatase (ASPP) from Escherichia coli either in the chloroplast or in the cytosol. Source leaves from plants expressing ASPP in the chloroplast exhibited reduced starch and normal ADPG content as compared with control plants. Most importantly however, leaves from plants expressing ASPP in the cytosol showed a large reduction of the levels of both ADPG and starch, whereas hexose phosphates increased as compared with control plants. No pleiotropic changes in photosynthetic parameters and maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism could be detected in the leaves expressing ASPP in the cytosol. The overall results show that, essentially similar to cereal endosperms, most of the ADPG linked to starch biosynthesis in source leaves occurs in the cytosol.Publication Open Access Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli(National Academy of Sciences, 2001) Moreno Bruna, Beatriz; Baroja Fernández, Edurne; Muñoz Pérez, Francisco José; Bastarrica Berasategui, Ainara; Zandueta Criado, Aitor; Rodríguez López, Milagros; Lasa Uzcudun, Íñigo; Akazawa, Takashi; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaAn adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as ‘‘nudix’’ hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wildtype bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli.