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1. A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

Author

Ana Cristina Ebrecht, Sergio Adrian Guerrero, Lucila Inés Martínez, Miguel A. Ballicora, Alberto A. Iglesias, Mabel Cristina Aleanzi, and Matías Damián Asención Diez

Subjects
Models, Molecular, Glucose-1-Phosphate Adenylyltransferase, medicine.disease_cause, purl.org/becyt/ford/1 [https], Streptococcus mutans, lcsh:Chemistry, Cloning, Molecular, protein engineering, allosteric regulation, pyrophosphorylases evolution, UDP-glucose, ADP-glucose, lcsh:QH301-705.5, Spectroscopy, chemistry.chemical_classification, 0303 health sciences, UTP—glucose-1-phosphate uridylyltransferase, Effector, 030302 biochemistry & molecular biology, General Medicine, Oligosaccharide, Bioquímica y Biología Molecular, Computer Science Applications, Biochemistry, lipids (amino acids, peptides, and proteins), CIENCIAS NATURALES Y EXACTAS, UTP-Glucose-1-Phosphate Uridylyltransferase, Recombinant Fusion Proteins, Allosteric regulation, Molecular Sequence Data, macromolecular substances, Biology, Catalysis, Article, Inorganic Chemistry, Ciencias Biológicas, 03 medical and health sciences, medicine, Escherichia coli, Amino Acid Sequence, Physical and Theoretical Chemistry, purl.org/becyt/ford/1.6 [https], Molecular Biology, 030304 developmental biology, Organic Chemistry, Glucosephosphates, Protein engineering, Fusion protein, carbohydrates (lipids), Enzyme, chemistry, lcsh:Biology (General), lcsh:QD1-999
Abstract

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. Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina; Fil: Ballicora, Miguel A.. University Of Chicago; Estados Unidos de América; Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina

Published
2013

2. Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

Author

Sergio Adrian Guerrero, Sergio Garay, Claudia Vanesa Piattoni, Lucila Inés Martínez, Daniel Rodrigues, and Alberto A. Iglesias

Subjects
Models, Molecular, UTP-Glucose-1-Phosphate Uridylyltransferase, UDP-glucose pyrophosphorylase, Protein Conformation, Intracellular Space, Carbohydrate metabolism, Biochemistry, Dithiothreitol, purl.org/becyt/ford/1 [https], Redox, Ciencias Biológicas, chemistry.chemical_compound, Entamoeba histolytica, Enzyme inducer, Cloning, Molecular, purl.org/becyt/ford/1.6 [https], chemistry.chemical_classification, Methionine, biology, General Medicine, Bioquímica y Biología Molecular, biology.organism_classification, Molecular biology, Enzyme assay, Recombinant Proteins, Enzyme, chemistry, Mutation, biology.protein, Mutagenesis, Site-Directed, Thioredoxin, Oxidation-Reduction, Protein Processing, Post-Translational, CIENCIAS NATURALES Y EXACTAS, Cysteine
Abstract

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. Fil: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina Fil: Piattoni, Claudia Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina Fil: Garay, Alberto. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Rodrigues, Daniel Enrique. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina

Published
2010

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