On the simultaneous activation of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by pyruvate and fructose 6-phosphate.

Bacterial ADP-glucose pyrophosphorylases are allosterically regulated by metabolites that are key intermediates of central pathways in the respective microorganism. Pyruvate (Pyr) and fructose 6-phosphate (Fru6P) activate the enzyme from Agrobacterium tumefaciens by increasing Vmax about 10- and 20-fold, respectively. Here, we studied the combined effect of both metabolites on the enzyme activation. Our results support a model in which there is a synergistic binding of these two activators to two distinct sites and that each activator leads the enzyme to distinct active forms with different properties. In presence of both activators, Pyr had a catalytically dominant effect over Fru6P determining the active conformational state. By mutagenesis we obtained enzyme variants still sensitive to Pyr activation, but in which the allosteric signal by Fru6P was disrupted. This indicated that the activation mechanism for each effector was not the same. The ability for this enzyme to have more than one allosteric activator site, active forms, and allosteric signaling mechanisms is critical to expand the evolvability of its regulation. These synergistic interactions between allosteric activators may represent a feature in other allosteric enzymes.

A novel dual allosteric activation mechanism of Escherichia coli ADP-glucose pyrophosphorylase: the role of pyruvate.

Fructose-1,6-bisphosphate activates ADP-glucose pyrophosphorylase and the synthesis of glycogen in Escherichia coli. Here, we show that although pyruvate is a weak activator by itself, it synergically enhances the fructose-1,6-bisphosphate activation. They increase the enzyme affinity for each other, and the combination increases Vmax, substrate apparent affinity, and decreases AMP inhibition. Our results indicate that there are two distinct interacting allosteric sites for activation. Hence, pyruvate modulates E. coli glycogen metabolism by orchestrating a functional network of allosteric regulators. We postulate that this novel dual activator mechanism increases the evolvability of ADP-glucose pyrophosphorylase and its related metabolic control.

A Chimeric UDP-glucose pyrophosphorylase produced by protein engineering exhibits sensitivity to allosteric regulators.

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.

Insights into glycogen metabolism in chemolithoautotrophic bacteria from distinctive kinetic and regulatory properties of ADP-glucose pyrophosphorylase from Nitrosomonas europaea.

Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO(2) via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function.

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.

Understanding the allosteric trigger for the fructose-1,6-bisphosphate regulation of the ADP-glucose pyrophosphorylase from Escherichia coli.

ADP-glucose pyrophosphorylase is the enzyme responsible for the regulation of glycogen synthesis in bacteria. The enzyme N-terminal domain has a Rossmann-like fold with three neighbor loops facing the substrate ATP. In the Escherichia coli enzyme, one of those loops also faces the regulatory site containing Lys(39), a residue involved in binding of the allosteric activator fructose-1,6-bisphosphate and its analog pyridoxal-phosphate. The other two loops contain Trp(113) and Gln(74), respectively, which are highly conserved among all the ADP-glucose pyrophosphorylases. Molecular modeling of the E. coli enzyme showed that binding of ATP correlates with conformational changes of the latter two loops, going from an open to a closed (substrate-bound) form. Alanine mutants of Trp(113) or Gln(74) did not change apparent affinities for the substrates, but they became insensitive to activation by fructose-1,6-bisphosphate. By capillary electrophoresis we found that the mutant enzymes still bind fructose-1,6-bisphosphate, with similar affinity as the wild type enzyme. Since the mutations did not alter binding of the activator, they must have disrupted the communication between the regulatory and the substrate sites. This agrees with a regulatory mechanism where the interaction with the allosteric activator triggers conformational changes at the level of loops containing residues Trp(113) and Gln(74).

Bi-national and interdisciplinary course in enzyme engineering.

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.

Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis.

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 Leu(102) and Pro(103) 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.

Diagnostico serologico de la enfermedad celiaca: anticuerpos anti-pepticos de sintesis de gliadina y anti-transglutaminasa de tejido / Serological diagnosis of disease: anti-gliadin peptide antitibodies and tissue anti-transglutaminase

Los marcadores serológicos comúnmente utilizados en el diagnóstico de la enfermedad celíaca son los anticuerpos antigliadina (AG) y antiendomisio (AE). Recientemente (1997) se identificó a la transglutaminasa de tejido (tTG) como el principal autoantígeno de los anticuerpos AE. El objetivo de este trabajo fue determinar la sensibilidad y especificidad de testes de ELISA desarrollados en base a la utilización de estructuras moleculares definidas como antígenos de captura para los anticuerpos AG y AE. Como antígenos inmovilizados para los anticuerpos AG se ensayaron tres péptidos de sínteses correspondientes a la región amino terminal de la alfa gliadina y para los AE, la transglutaminasa de hígado de cobayo. Se examinaron un total de 80 sueros correspondientes a: pacientes celíacos, no tratados y tratados, controles enfermos no celíacos y controles sanos. Rango de edad: 7 meses a 14 años. Se obtuvo una sensibilidad del 97 por ciento y una especificidad 86 por ciento para la IgG determinada utilizando como antígeno uno de los tres péptidos de síntesis (correspondiente a los residuos 31-55 de la alfa gliadina). Este péptido aparece como un antígeno altamente sensible y más específico que la gliadina. El mejor resultado, con un 100 por ciento de especificidad y sensibilidad, se obtuvo en la determinación de la IgA anti-tTG, lo que destaca la relevancia de estos anticuerpos como marcadores serológicos de la enfermedad celíaca.

Diagnostico serologico de la enfermedad celiaca: anticuerpos anti-pepticos de sintesis de gliadina y anti-transglutaminasa de tejido / Serological diagnosis of disease: anti-gliadin peptide antitibodies and tissue anti-transglutaminase

Los marcadores serológicos comúnmente utilizados en el diagnóstico de la enfermedad celíaca son los anticuerpos antigliadina (AG) y antiendomisio (AE). Recientemente (1997) se identificó a la transglutaminasa de tejido (tTG) como el principal autoantígeno de los anticuerpos AE. El objetivo de este trabajo fue determinar la sensibilidad y especificidad de testes de ELISA desarrollados en base a la utilización de estructuras moleculares definidas como antígenos de captura para los anticuerpos AG y AE. Como antígenos inmovilizados para los anticuerpos AG se ensayaron tres péptidos de sínteses correspondientes a la región amino terminal de la alfa gliadina y para los AE, la transglutaminasa de hígado de cobayo. Se examinaron un total de 80 sueros correspondientes a: pacientes celíacos, no tratados y tratados, controles enfermos no celíacos y controles sanos. Rango de edad: 7 meses a 14 años. Se obtuvo una sensibilidad del 97 por ciento y una especificidad 86 por ciento para la IgG determinada utilizando como antígeno uno de los tres péptidos de síntesis (correspondiente a los residuos 31-55 de la alfa gliadina). Este péptido aparece como un antígeno altamente sensible y más específico que la gliadina. El mejor resultado, con un 100 por ciento de especificidad y sensibilidad, se obtuvo en la determinación de la IgA anti-tTG, lo que destaca la relevancia de estos anticuerpos como marcadores serológicos de la enfermedad celíaca. (AU)