RESUMO
Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.
Assuntos
Metabolismo dos Carboidratos , Euglena gracilis/enzimologia , Glucanos/química , UTP-Glucose-1-Fosfato Uridililtransferase/química , Catálise , Glucanos/metabolismo , Cinética , Domínios Proteicos , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.
Assuntos
Membrana Celular/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/biossíntese , Caules de Planta/enzimologia , Saccharum/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/biossíntese , Membrana Celular/química , Modelos Moleculares , Fosforilação/fisiologia , Proteínas de Plantas/química , Caules de Planta/química , Estrutura Terciária de Proteína , UTP-Glucose-1-Fosfato Uridililtransferase/química , Uridina Difosfato Glucose/biossíntese , Uridina Difosfato Glucose/químicaRESUMO
In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.
Assuntos
Escherichia coli/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Streptococcus mutans/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/genética , Glucofosfatos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Streptococcus mutans/química , Streptococcus mutans/genética , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V(max) values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.
Assuntos
Entamoeba histolytica/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Clonagem Molecular , Espaço Intracelular/enzimologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60kDa, exhibiting relatively high specific activity ( approximately 80Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15microM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term.