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.

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 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.

Characterization of recombinant UDP- and ADP-glucose pyrophosphorylases and glycogen synthase to elucidate glucose-1-phosphate partitioning into oligo- and polysaccharides in Streptomyces coelicolor.

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.

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).

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)