On the kinetic and allosteric regulatory properties of the ADP-glucose pyrophosphorylase from Rhodococcus jostii: An approach to evaluate glycogen metabolism in oleaginous bacteria

Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.Fil: Cereijo, Antonela Estefanía. 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; ArgentinaFil: Asención Diez, Matías Damián. 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; ArgentinaFil: Dávila Costa, José Sebastián. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia ; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia Austral. Centro de Investigaciones y Transferencia Golfo San Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia Golfo San Jorge. Universidad Nacional de la Patagonia ; ArgentinaFil: 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; Argentin

Structure, function, and evolution of plant ADP-glucose pyrophosphorylase

Key message: This review outlines research performed in the last two decades on the structural, kinetic,regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzymefor starch biosynthesis. Abstract: ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.Fil: 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; ArgentinaFil: Asención Diez, Matías Damián. 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; ArgentinaFil: Ballicora, Miguel A.. Loyola University Maryland (lum);Fil: 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; Argentin

Iodine Staining of Escherichia coli Expressing Genes Involved in the Synthesis of Bacterial Glycogen

The presence of intracellular glycogen can be detected by the following iodine staining technique. Cells with glycogen stain dark brown, whereas in its absence they remain with a pale yellowish color. It is hypothesized that iodine atoms fit into helical coils formed by the α-polyglucan to form a coloured glycogen-iodine complex. Here, we have studied the expression of Streptococcus mutans (S. mutans) genes that control the biosynthesis of this polysaccharide. Thus, we expressed glgC and glgD genes coding for both ADP-Glc pyrophosphorylase subunits in Escherichia coli (E. coli) AC70R1-504 cells to complement the deficient accumulation of glycogen by this strain. In control cells or in those where an inactive protein was expressed, the synthesis of the polysaccharide was undetectable by this iodine staining technique.Fil: Demonte, Ana M.. Universidad Nacional del Litoral; ArgentinaFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Chicago. Department of Chemistry & Biochemistry; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; Argentin

The Crystal Structure of Nitrosomonas Europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into the Sucrose Metabolism in Prokaryotes

In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the non-photosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure from the plant Arabidopsis thaliana was in a closed conformation. Comparative structural analysis revealed a “hinge-latch” combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family showed to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family, but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded

Structural analysis reveals a pyruvate-binding activator site in the Agrobacterium tumefaciens ADP–glucose pyrophosphorylase

The pathways for biosynthesis of glycogen inbacteria and starch in plants are evolutionarily andbiochemically related. They are regulated primarily by ADP?glucose pyrophosphorylase, which evolved to satisfy metabolic requirements of a particular organism. Despite the importance of these two pathways, little is known about the mechanism that controls pyrophosphorylase activity or the location of its allosteric sites. Here, we report pyruvate-bound crystal structures of ADP-glucose pyrophosphorylase from the bacterium Agrobacterium tumefaciens, identifying a previously elusive activator site for the enzyme. We found that the tetrameric enzyme binds two molecules of pyruvate in a planar conformation. Each binding site is located in a crevice between the C-terminal domains of two subunits where they stack via a distinct β-helix region. Pyruvate interacts with the side chain of Lys-43 and with the peptide backbone of Ser-328 and Gly-329 from both subunits. These structural insights led to the design of two variants with altered regulator properties. In one variant (K43A), the allosteric effect was absent, whereas in the other (G329D), the introduced Asp mimicked the presence of pyruvate. The latter generated an enzyme that was pre-activated and insensitive to further activation by pyruvate. Our study furnishes a deeper understanding of how glycogen biosynthesis is regulated in bacteria and the mechanism by which transgenic plants increased their starch production. These insights will facilitate rational approaches to enzyme engineering for starch production in crops of agricultural interest and will promote further study of allosteric signal transmission and molecular evolution in this important enzyme family.Fil: Hill, B. L.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Mascarenhas, R.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Patel, H. P.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Asención Diez, Matías Damián. 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; ArgentinaFil: Wu, R.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: 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; ArgentinaFil: Liu, D.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados UnidosFil: Ballicora, M. A.. Dpt Of Chem And Biochemistry. Loyola University Chicago; Estados Unido

Developmental delay in a Streptomyces venezuelae glgE null mutant is associated with the accumulation of alpha-maltose 1-phosphate

The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like alpha-glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give alpha-maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend alpha-1,4-linked maltooligosaccharides, which are thought to be branched with alpha-1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of a-glucan deposition. A constructed Delta glgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated alpha-maltose 1-phosphate and maltose but no alpha-glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the Delta glgE mutant on galactose and that of a Delta pep2 mutant on maltose were analysed. In both cases, neither accumulation of alpha-maltose 1-phosphate/alpha-glucan nor a developmental delay was observed. Thus, high levels of alpha-maltose 1-phosphate are responsible for the developmental phenotype of the Delta glgE mutant, rather than the lack of a-glucan

Glucosamine-P and rhodococcal ADP-glucose pyrophosphorylases: A hint to (re)discover (actino)bacterial amino sugar metabolism

Glycogen was described as a temporal storage molecule in rhodococci, interconnecting lipids and carbon availability. The Rhodococcus jostii ADP-glucose pyrophosphorylase (ADP-GlcPPase) kinetic and regulatory properties support this role. Curiously, the enzyme uses glucosamine-1P as alternative substrate. Herein, we report the in-depth study of glucosamine-1P activity and its regulation in two rhodocoocal ADP-GlcPPases, finding that glucosamine-6P (representing a metabolic carbon/nitrogen node) is a critical activator, then reinforcing the role of glycogen as an ?intermediary metabolite? in rhodococci. Glucosamine-1P activity in rhodococcal ADP-GlcPPases responds to activation by metabolites improving their catalytic performance, strongly suggesting its metabolic feasibility. This work supports a scenario for new molecules/metabolites discovery and hypothesizes on evolutionary mechanisms underlying enzyme promiscuity opening novel metabolic features in (actino)bacteria.Fil: Cereijo, Antonela Estefanía. 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; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia "San Juan Bosco"; ArgentinaFil: 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; ArgentinaFil: Asención Diez, Matías Damián. 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; Argentin

Regulatory properties of the ADP-glucose pyrophosphorylase from the clostridial Firmicutes member Ruminococcus albus

ADP-glucose pyrophosphorylase from Firmicutes is encoded by two genes (glgC and glgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups of Firmicutes, Bacillales and Lactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase from Clostridiales, the third group in Firmicutes, has been characterized. For this reason, we cloned the glgC and glgD genes from Ruminococcus albus. Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of the Bacillales enzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases from Firmicutes were unregulated. Our results, together with data from Lactobacillales, indicate that heterotetrameric Firmicutes enzymes are mostly regulated. Thus, the ADPglucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation.Fil: Cereijo, Antonela Estefanía. 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; ArgentinaFil: Asención Diez, Matías Damián. 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; ArgentinaFil: Ballicora, Miguel A.. University of Chicago; Estados UnidosFil: 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; Argentin

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