Authors: Asención Diez, Matías Damián; Ebrecht, Ana Cristina; Martínez, Lucila Inés; Aleanzi, Mabel Cristina; Guerrero, Sergio Adrian; Ballicora, Miguel A.; Iglesias, Alberto Alvaro
Publication Date: 2013.
Language: English.
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.
Author affiliation: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Instituto de Agrobiotecnologia del Litoral; Argentina;
Author affiliation: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Author affiliation: Martínez, Lucila Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Author affiliation: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Author affiliation: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Author affiliation: Ballicora, Miguel A.. University Of Chicago; Estados Unidos de América;
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Publication Date: 2003.
Language: English.
Abstract:
ADP-Glucose Pyrophosphorylase, a Regulatory Enzyme forBacterial Glycogen SynthesisMiguel A. Ballicora,1 Alberto A. Iglesias,2 and Jack Preiss1*Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824,1and Bioquímica Ba´sica de Macromole´culas, Grupo de Enzimologı´a Molecular, Facultad de Bioquı´mica yCiencias Biolo´gicas, Universidad Nacional del Litoral, Santa Fe, Argentina2INTRODUCTION: FUNCTION AND REGULATION OF ADP-Glc PPase INBACTERIA AND IN PLANTS ......................................................................213PHYSIOLOGICAL ROLE..............................................................................214REGULATORY PROPERTIES AND QUATERNARY STRUCTURE OFADP-Glc PPases FROM DIFFERENT SOURCES.........................................214SUBUNIT STRUCTURE OF PLANT ENZYMES ........................................216IDENTIFICATION OF IMPORTANT AMINO ACID RESIDUES...............216PREDICTION OF THE STRUCTURE OF ADP-Glc PPases .........................217CATALYTIC RESIDUES.......................................................................................................221DOMAIN CHARACTERIZATION.................................................................222EVOLUTION OF ADP-Glc PPases.................................................................222ACKNOWLEDGMENTS ...............................................................................223REFERENCES.................................................................................................223
Author affiliation: Ballicora, Miguel. Michigan State University; Estados Unidos
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Preiss, Jack. Michigan State University; Estados Unidos
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Authors: Giordano, Pablo César; Martínez, Hugo D.; Iglesias, Alberto Alvaro; Beccaria, Alejandro José; Goicoechea, Hector Casimiro
Publication Date: 2010.
Language: English.
Abstract:
Production of recombinant Oryza sativa non-symbiotic hemoglobin 1 (OsHb1) by Escherichia coli was maximized in shake-flask cultures in media containing tryptone, yeast extract, sodium chloride and byproduct glycerol from biodiesel production. Response surface methodology (RSM) and artificial neural networks (ANNs), followed by multiple response optimization through a desirability function were applied to evaluate the amount of OsHb1 produced. The results obtained by the application of ANNs were more reliable since better statistical parameters were obtained. The optimal conditions were (g L-1), tryptone, 42.69; yeast extract, 20.11; sodium chloride, 17.77; and byproduct glycerol, 0.33. A maximum recombinant protein concentration of 3.50 g L-1 and a minimum biomass concentration of 18.48 g L -1 were obtained under these conditions. Although the concentrations of tryptone, yeast extract and sodium chloride are relatively high, the increase in the yield with respect to biomass formed (YP/X) overcomes this disadvantage.
Author affiliation: Giordano, Pablo César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Martínez, Hugo D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Beccaria, Alejandro José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Goicoechea, Hector Casimiro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Keywords: ARTIFICIAL NEURAL NETWORKS; BYPRODUCT GLYCEROL; ESCHERICHIA COLI; RECOMBINANT PROTEIN; RESPONSE SURFACE METHODOLOGY; Química Analítica; Ciencias Químicas; CIENCIAS NATURALES Y EXACTAS; Bioprocesamiento Tecnológico, Biocatálisis, Fermentación; Biotecnología Industrial; INGENIERÍAS Y TECNOLOGÍAS.
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Authors: Kuhn, Misty L.; Figueroa, Carlos Maria; Aleanzi, Mabel Cristina; Olsen, Kenneth W.; Iglesias, Alberto Alvaro; Ballicora, Miguel A.
Publication Date: 2010.
Language: English.
Abstract:
Higher education institutions and scientific funding agencies are emphasizing international projects that involve the integration and synergy between research groups, particularly if different disciplines are involved. Students with an education that reflects these trends will have more tools to succeed in the future, but it is challenging to provide this type of learning experience. Here we present the organization of a bi-national course with the goals to teach students protein structure/function relationships, which give them actual research experience in both computational and experimental laboratories, and engage them in an international networking experience. Two collaborative learning courses were organized at Loyola University Chicago (USA) and Universidad Nacional del Litoral (Argentina) for graduate and advanced undergraduate students. Multiple instructors at different stages in their careers gave lectures during the course and were able to interact with students on a one-on-one basis. Nearly every student from both institutions thoroughly enjoyed this approach, and they learned more about protein structure and gained important tools for their own research. We believe that this type of course design is applicable and transferable to other institutions and areas of science. We found that the combination of international networking and incorporation of actual research projects ignited the enthusiasm of students and instructors. Due to the success of these courses, we planned to incorporate them as regular series in our curriculum.
Author affiliation: Kuhn, Misty L.. Loyola University Chicago; Estados Unidos
Author affiliation: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Olsen, Kenneth W.. Loyola University Chicago; Estados Unidos
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Ballicora, Miguel A.. Loyola University Chicago; Estados Unidos
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Authors: Barchiesi, Julieta; Hedin, Nicolas; Iglesias, Alberto Alvaro; Gomez Casati, Diego Fabian; Ballicora, Miguel A.; Busi, María Victoria
Publication Date: 2017.
Language: English.
Abstract:
Hydrosoluble glycogen is the major energy storage compound in bacteria, archaea, fungi, and animal cells. In contrast, photosynthetic eukaryotes have evolved to build a highly organized semicrystalline granule of starch. Several enzymes are involved in polysaccharide synthesis, among which glycogen or starch synthase catalyze the elongation of the α-1,4-glucan chain. Ostreococcus tauri, accumulates a single starch granule and contains three starch synthase III (SSIII) isoforms, known as OsttaSSIII-A, OsttaSSIII-B and OsttaSSIII-C. After amino acids sequence analysis we found that OsttaSSIII-C lacks starch-binding domains, being 49% identical to the catalytic region of the SSIII from Arabidopsis thaliana and 32% identical to the entire Escherichia coli glycogen synthase. The recombinant, highly purified OsttaSSIII-C exhibited preference to use as a primer branched glycans (such as rabbit muscle glycogen and amylopectin), rather than amylose. Also, the enzyme displayed a high affinity toward ADP-glucose. We found a marked conservation of the amino acids located in the catalytic site, and specifically determined the role of residues R270, K275 and E352 by site-directed mutagenesis. Results show that these residues are important for OsttaSSIII-C activity, suggesting a strong similarity between the active site of the O. tauri SSIII-C isoform and other bacterial glycogen synthases.
Author affiliation: Barchiesi, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina
Author affiliation: Hedin, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina
Author affiliation: Ballicora, Miguel A.. Loyola University Chicago; Estados Unidos
Author affiliation: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosinteticos y Bioquimicos. Universidad Nacional de Rosario. Facultad de Cs.bioquímicas y Farmaceuticas. Centro de Estudios Fotosinteticos y Bioquimicos; Argentina
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Authors: Barchiesi, Julieta; Hedin, Nicolas; Iglesias, Alberto Alvaro; Gomez Casati, Diego Fabian; Ballicora, Miguel A.; Busi, María Victoria
Publication Date: 2017.
Language: English.
Abstract:
Hydrosoluble glycogen is the major energy storage compound in bacteria, archaea, fungi, and animal cells. In contrast, photosynthetic eukaryotes have evolved to build a highly organized semicrystalline granule of starch. Several enzymes are involved in polysaccharide synthesis, among which glycogen or starch synthase catalyze the elongation of the α-1,4-glucan chain. Ostreococcus tauri, accumulates a single starch granule and contains three starch synthase III (SSIII) isoforms, known as OsttaSSIII-A, OsttaSSIII-B and OsttaSSIII-C. After amino acids sequence analysis we found that OsttaSSIII-C lacks starch-binding domains, being 49% identical to the catalytic region of the SSIII from Arabidopsis thaliana and 32% identical to the entire Escherichia coli glycogen synthase. The recombinant, highly purified OsttaSSIII-C exhibited preference to use as a primer branched glycans (such as rabbit muscle glycogen and amylopectin), rather than amylose. Also, the enzyme displayed a high affinity toward ADP-glucose. We found a marked conservation of the amino acids located in the catalytic site, and specifically determined the role of residues R270, K275 and E352 by site-directed mutagenesis. Results show that these residues are important for OsttaSSIII-C activity, suggesting a strong similarity between the active site of the O. tauri SSIII-C isoform and other bacterial glycogen synthases.
Author affiliation: Barchiesi, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina
Author affiliation: Hedin, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina
Author affiliation: Ballicora, Miguel A.. Loyola University Chicago; Estados Unidos
Author affiliation: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Publication Date: 2008.
Language: English.
Abstract:
Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Because non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (NP-Ga3PDHase) is involved in the production of reductive power (NADPH) in the cytosol, its behavior under oxidative stress conditions was analyzed. The specific activity of the enzyme was found to increase up to 2-fold after oxidative conditions imposed by methylviologen in wheat and maize seedlings. Under moderate oxidant concentration, lack of mRNA induction was observed. The increase in specific activity would thus be a consequence of a significant stability of NP-Ga3PDHase. Our results suggest that the enzyme could be modified by oxidation of cysteine residues, but formation of disulfide bridges is dependent on levels of divalent cations and 14-3-3 proteins. The latter differential effect could be critical to relatively maintain energy and reductant levels in the cytoplasm of plant cells under oxidative stress.
Author affiliation: Bustos, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Author affiliation: Bustamante, Claudia Anabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas
Authors: Cabeza, Matías Sebastián; Guerrero, Sergio Adrian; Iglesias, Alberto Alvaro; Arias, Diego Gustavo
Publication Date: 2015.
Language: English.
Abstract:
Background Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified. Methods The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica. Results We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin. Conclusions The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species. General significance To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins.
Author affiliation: Cabeza, Matías Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Author affiliation: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Repository: CONICET Digital (CONICET). Consejo Nacional de Investigaciones Científicas y Técnicas