Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.

Carbohydrate Metabolism in Bacteria: Alternative Specificities in ADP-Glucose Pyrophosphorylases Open Novel Metabolic Scenarios and Biotechnological Tools.

We explored the ability of ADP-glucose pyrophosphorylase (ADP-Glc PPase) from different bacteria to use glucosamine (GlcN) metabolites as a substrate or allosteric effectors. The enzyme from the actinobacteria Kocuria rhizophila exhibited marked and distinctive sensitivity to allosteric activation by GlcN-6P when producing ADP-Glc from glucose-1-phosphate (Glc-1P) and ATP. This behavior is also seen in the enzyme from Rhodococcus spp., the only one known so far to portray this activation. GlcN-6P had a more modest effect on the enzyme from other Actinobacteria (Streptomyces coelicolor), Firmicutes (Ruminococcus albus), and Proteobacteria (Agrobacterium tumefaciens) groups. In addition, we studied the catalytic capacity of ADP-Glc PPases from the different sources using GlcN-1P as a substrate when assayed in the presence of their respective allosteric activators. In all cases, the catalytic efficiency of Glc-1P was 1-2 orders of magnitude higher than GlcN-1P, except for the unregulated heterotetrameric protein (GlgC/GgD) from Geobacillus stearothermophilus. The Glc-1P substrate preference is explained using a model of ADP-Glc PPase from A. tumefaciens based on the crystallographic structure of the enzyme from potato tuber. The substrate-binding domain localizes near the N-terminal of an α-helix, which has a partial positive charge, thus favoring the interaction with a hydroxyl rather than a charged primary amine group. Results support the scenario where the ability of ADP-Glc PPases to use GlcN-1P as an alternative occurred during evolution despite the enzyme being selected to use Glc-1P and ATP for α-glucans synthesis. As an associated consequence in such a process, certain bacteria could have improved their ability to metabolize GlcN. The work also provides insights in designing molecular tools for producing oligo and polysaccharides with amino moieties.

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 enzyme for starch biosynthesis. 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.

The ADP-glucose pyrophosphorylase from Melainabacteria: a comparative study between photosynthetic and non-photosynthetic bacterial sources.

Until recently, the cyanobacterial phylum only included oxygenic photosynthesizer members. The discovery of Melainabacteria as a group of supposed non-photosynthetic cyanobacteria asked to revisit such scenario. From metagenomic data, we were able to identify sequences encoding putative ADP-glucose pyrophosphorylases (ADP-GlcPPase) from free-living and intestinal Melainabacteria. The respective genes were de novo synthesized and over-expressed in Escherichia coli. The purified recombinant proteins from both Melainabacteria species were active as ADP-GlcPPases, exhibiting Vmax values of 2.3 (free-living) and 7.1 U/mg (intestinal). The enzymes showed similar S0.5 values (∼0.3 mM) for ATP, while the one from the intestinal source exhibited a 6-fold higher affinity toward glucose-1P. Both recombinant ADP-GlcPPases were sensitive to glucose-6P activation (A0.5 ∼0.3 mM) and Pi and ADP inhibition (I0.5 between 0.2 and 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. As far as we know, this is the first biochemical characterization of an active enzyme from Melainabacteria. This work contributes to a better understanding of the evolution of allosteric regulation in the ADP-GlcPPase family, which is critical for synthesizing the main reserve polysaccharide in prokaryotes (glycogen) and plants (starch). In addition, our results offer further information to discussions regarding the phylogenetic position of Melainabacteria.

Phosphorylation of ADP-Glucose Pyrophosphorylase During Wheat Seeds Development.

Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro, the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination.

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.

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.

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

The pathways for biosynthesis of glycogen in bacteria and starch in plants are evolutionarily and biochemically 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 ß-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 regulatory 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 preactivated 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.

On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits.

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

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 ADP-glucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation.IMPORTANCE The enzymes involved in glycogen synthesis from Firmicutes have been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase from Ruminococcus albus Our results showed that this protein that belongs to different groups from Firmicutes (Bacillales, Lactobacillales, and Clostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in the Firmicutes group, whereby we propose that these heterotetrameric enzymes, with the exception of Bacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family in Firmicutes.

On the stability of nucleoside diphosphate glucose metabolites: implications for studies of plant carbohydrate metabolism.

Nucleoside diphosphate sugars (NDP-sugars) are the substrates for biosynthesis of oligo- and polysaccharides, such as starch and cellulose, and are also required for biosynthesis of nucleotides, ascorbic acid, several cofactors, glycoproteins and many secondary metabolites. A controversial study that questions the generally accepted pathway of ADP-glucose and starch synthesis in plants is based, in part, on claims that NDP-sugars are unstable at alkaline pH in the presence of Mg2+ and that this instability can lead to unreliable results from in vitro assays of enzyme activities. If substantiated, this claim would have far-reaching implications for many published studies that report on the activities of NDP-sugar metabolizing enzymes. To resolve this controversy, we investigated the stability of UDP- and ADP-glucose using biophysical, namely nuclear magnetic resonance (NMR), and highly specific enzymatic methods. Results obtained with both techniques indicate that NDP-sugars are not as unstable as previously suggested. Moreover, their calculated in vitro half-lives are significantly higher than estimates of their in planta turnover times. This indicates that the physico-chemical stability of NDP-sugars has little impact on their concentrations in vivo and that NDP-sugar levels are determined primarily by the relative rates of enzymatic synthesis and consumption. Our results refute one of the main arguments for the controversial pathway of starch synthesis from imported ADP-glucose produced by sucrose synthase in the cytosol.

Monofluorophosphate Blocks Internal Polysaccharide Synthesis in Streptococcus mutans.

Streptococcus mutans is the leading cause of dental caries worldwide by accumulating a glycogen-like internal polysaccharide (IPS) that contributes to cariogenicity when sugars are in excess. Sodium monofluorophosphate (MFP) is an active anticariogenic compound in toothpastes. Herein, we show that MFP inhibits (with an I0.5 of 1.5 mM) the S. mutans ADP-glucose pyrophosphorylase (EC 2.7.7.27), which catalyzes the key step in IPS biosynthesis. Enzyme inhibition by MFP is similar to orthophosphate (Pi), except that the effect caused by MFP is not reverted by fructose-1,6-bisP, as occurs with Pi. Inhibition was correlated with a decrease in acidogenesis and IPS accumulation in S. mutans cells cultured with 2 mM sodium MFP. These effects were not mimicked by sodium fluoride. Considering that glycogen synthesis occurs by different pathways in mammals and bacteria, ADP-glucose pyrophosphorylase could be visualized as a molecular target for controlling S. mutans virulence. Our results strongly suggest that MFP is a suitable compound to affect such a target, inducing an anticariogenic effect primarily by inhibiting a key step in IPS synthesis.

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate.

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.

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

UNLABELLED: In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the nonphotosynthetic 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 was shown to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, and 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. IMPORTANCE: We obtained biochemical and structural evidence of sucrose metabolism in nonphotosynthetic bacteria. Until now, only sucrose synthases from photosynthetic organisms have been characterized. Here, we provide the crystal structure of the sucrose synthase from the chemolithoautotroph N. europaea. The structure supported that the enzyme functions with an open/close induced fit mechanism. The enzyme prefers as the substrate adenine-based nucleotides rather than uridine-based like the eukaryotic counterparts, implying a strong connection between sucrose and glycogen metabolism in these bacteria. Mutagenesis data showed that the catalytic mechanism must be conserved not only in sucrose synthases but also in all other retaining GT-B glycosyltransferases.

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. RESULTS: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. CONCLUSIONS: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. GENERAL SIGNIFICANCE: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate.

BACKGROUND: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes. RESULTS: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification. CONCLUSIONS: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides. GENERAL SIGNIFICANCE: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.

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.

The ADP-glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes.

Streptococcus mutans is the leading cause of dental caries worldwide. The bacterium accumulates a glycogen-like internal polysaccharide, which mainly contributes to its carionegic capacity. S.mutans has two genes (glgC and glgD) respectively encoding putative ADP-glucose pyrophosphorylases (ADP-Glc PPase), a key enzyme for glycogen synthesis in most bacteria. Herein, we report the molecular cloning and recombinant expression of both genes (separately or together) followed by the characterization of the respective enzymes. When expressed individually GlgC had ADP-Glc PPase activity, whereas GlgD was inactive. Interestingly, the coexpressed GlgC/GlgD protein was one order of magnitude more active than GlgC alone. Kinetic characterization of GlgC and GlgC/GlgD pointed out remarkable differences between them. Fructose-1,6-bis-phosphate activated GlgC by twofold, but had no effect on GlgC/GlgD. Conversely, phospho-enol-pyruvate and inorganic salts inhibited GlgC/GlgD without affecting GlgC. However, in the presence of fructose-1,6-bis-phosphate GlgC acquired a GlgC/GlgD-like behaviour, becoming sensitive to the stated inhibitors. Results indicate that S. mutans ADP-Glc PPase is an allosteric regulatory enzyme exhibiting sensitivity to modulation by key intermediates of carbohydrates metabolism in the cell. The particular regulatory properties of the S.mutans enzyme agree with phylogenetic analysis, where GlgC and GlgD proteins found in other Firmicutes arrange in distinctive clusters.

The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus.

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. In filamentous cyanobacteria, the sucrose cleavage direction plays a key physiological function in carbon metabolism, nitrogen fixation, and stress tolerance. In unicellular strains, the function of sucrose synthase has not been elucidated. We report a detailed biochemical characterization of sucrose synthase from Thermosynechococcus elongatus after the gene was artificially synthesized for optimal expression in Escherichia coli. The homogeneous recombinant sucrose synthase was highly specific for ADP as substrate, constituting the first one with this unique characteristic, and strongly suggesting an interaction between sucrose and glycogen metabolism.