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

Abstract:

In plants, the synthesis of starch occurs by utilizing ADP-glucose as the glucosyl donor for the elongation of α-1,4-glucosidic chains. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway. The first committed step in these pathways is the synthesis of ADP-glucose in a reaction catalyzed by ADP-glucose pyrophosphorylase (ADPG1c PPase). Generally, this enzyme is allosterically regulated by intermediates of the major 1 carbon assimilatory pathway in the respective organism. In oxygenic photosynthesizers, ADPG1c PPase is mainly regulated by 3-phosphoglycerate (activator) and inorganic orthophosphate (inhibitor), interacting in four different patterns. Recent reports have shown that in higher plants, some of the enzymes could also be redox regulated. In eukaryotes, the enzyme is a heterotetramer comprised of two distinct subunits, a catalytic and a modulatory subunit. The latter has been proposed as related to variations in regulation of the enzyme in different plant tissues. Random and site-directed mutagenesis experiments of conserved amino acids revealed important residues for catalysis and regulation. Prediction of the ADPG1c PPase secondary structure suggests that it shares a common folding pattern to other sugar-nucleotide pyrophosphorylases, and they evolved from a common ancestor.

Author affiliation: Ballicora, Miguel A.. 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

Abstract:

Adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5′-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [ 14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[ 14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S0.5 determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.

Author affiliation: Yep, Alejandra. Michigan State University; Estados Unidos

Author affiliation: Bejar, Clarisa M.. Michigan State University; Estados Unidos

Author affiliation: Ballicora, Miguel A.. Michigan State University; Estados Unidos

Author affiliation: Dubay, Jennifer R.. 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

Abstract:

ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate. Little is known about the regions determining the specificity for the allosteric regulator. To study the function of different domains, two chimeric enzymes were constructed. "AE" contains the N-terminus (271 amino acids) of the A. tumefaciens ADPglucose pyrophosphorylase and the C-terminus (153 residues) of the E. coli enzyme, and "EA", the inverse construction. Expression of the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid. Characterization of the purified chimeric enzymes showed that the C-terminus of the E. coli enzyme is relevant for the selectivity by FBP. However, this region seems to be less important for the specificity by F6P in the A. tumefaciens enzyme. The chimeric enzyme AE is activated by both FBP and F6P, neither of which affect EA. Pyruvate activates EA with higher apparent affinity than AE, suggesting that the C-terminus of the A. tumefaciens enzyme plays a role in the binding of this effector. The allosteric inhibitor site is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes.

Author affiliation: Ballicora, Miguel A.. Michigan State University; Estados Unidos

Author affiliation: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; 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: Preiss, Jack. Michigan State University; Estados Unidos

Abstract:

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.

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 Of Chicago;

Author affiliation: Sesma, Juliana. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

Author affiliation: Preiss, Jack. Michigan State University; Estados Unidos

Abstract:

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu102 and Pro103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.

Author affiliation: Ballicora, Miguel A.. Loyola University; Estados Unidos

Author affiliation: Erben, Esteban Daniel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Yazaki, Terutaka. Michigan State University; Estados Unidos

Author affiliation: Bertolo, Ana L.. Michigan State University; Estados Unidos

Author affiliation: Demonte, Ana María Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina

Author affiliation: Schmidt, Jennifer R.. Michigan State University; Estados Unidos

Author affiliation: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina

Author affiliation: Bejar, Clarisa M.. Michigan State University; Estados Unidos

Author affiliation: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; Argentina

Author affiliation: Fusari, Corina M.. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Enzimología Molecular; 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

Author affiliation: Preiss, Jack. Michigan State University; Estados Unidos

Abstract:

The effect of temperature on the activity and stability of ADPglucose pyrophosphorylase from Anabaena PCC 7120 was studied. Experimental optima temperatures were found around 37-40°C or 42-45°C, depending on the absence or the presence of allosteric effectors in the assay medium, respectively. In the range of temperature where the enzyme is stable, curved Arrhenius plots were obtained, indicating a transition temperature between 9 and 12°C. Since these results were observed for both the forward and reverse reaction, with two different sets of substrates and two entirely different assay procedures, it seems unlikely that the effect can be on any component of the system other than the enzyme itself. Results suggest that cyanobacterial ADPglucose pyrophosphorylase undergoes conformational changes at different temperatures, rendering structures with different catalytic efficiencies. The different structures of the enzyme were visualized by emission fluorescence. ADPglucose pyrophosphorylase was irreversibly inactivated when exposed to temperatures above 40°C. Inactivation was dependent on temperature and followed first order kinetics. The substrate, ATP, and the allosteric effectors, 3PGA and Pi, effectively protected the enzyme against thermal inactivation. Protection afforded by ATP was affected by MgCl2. These results suggest that the binding of the effectors to the enzyme resulted in conformational changes of the protein, rendering structures more stable to temperature treatments. Similar structures could be adopted by the enzyme in different environments, since the higher stability was observed in media containing either high ionic strength or high hydrophobicity.

Author affiliation: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Author affiliation: Preiss, Jack. 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