Polysaccharides Biosynthesis in Streptococcus mutans: Characterization of ADP-glucose pyrophosphorylase

M.D. ASENCIÓN DIEZ; A.M. DEMONTE; S.A. GUERRERO; A.A. IGLESIAS

San Diego, CA

Congreso; 110th General Meeting American Society for Microbiology; 2010

American Society for Microbiology

Polysaccharides Biosynthesis in Streptococcus mutans: Characterization of ADP-glucose PyrophosphorylaseM. D. Asención Diez, A. M. M. Demonte, S. A. Guerrero, A. A. Iglesias; IAL, Univ. Natl. Litoral- CONICET, Santa Fe, ARGENTINA.Streptococcus mutans is the leading cause of dental caries worldwide. When growing in excess of sugar, the bacterium accumulates a glycogen-like polysaccharide (IPS); the polymer significantly contributing to cariogenicity. Despite this relevance, studies on IPS metabolism are scarce, this importantly restricting the understanding of the microorganism functioning and capacity at the molecular level. In this work, we present the molecular cloning of two genes, glgC and glgD, coding for ADP-glucose pyrophosphorylase (ADP-Glc PPase), a key enzyme for glycogen synthesis in most bacteria. Utilizing compatible plasmids, vectors were constructed for the expression of each geneseparately or together. The recombinant GlgC protein showed active as ADP-Glc PPase, whereas GlgD exhibited negligible activity. Interestingly, the joint expression of the genes glgC and glgD rendered a product having activity one order of magnitude higher than after the expression of glgC alone, thus suggesting the formation of a recombinant GlgCD protein form. Both active enzymes (GlgC and GlgCD) were purified and kinetically characterized. Fructose-1,6-bisphosphate was found to activate by near 2-to 4-fold GlgC and GlgCD, with a main effect in increasing the affinity of each enzyme toward the substrate ATP. Results suggest that synthesis of IPS in S. mutans could be regulated at the step catalyzed by ADP-Glc PPase. Two main mechanisms could be involved in such a regulation. One level of regulation could involve the formation of different oligomeric forms of the enzyme, which could comprise the dissimilar expression of each gene and/or the differential arrangement of oligomers. Another regulation could comprise the allosteric regulation of ADP-Glc PPase by intermediates of the glycolytic pathway. The whole picture supports a key involvement of the enzyme for the biochemistry of carbohydrates in S. mutans. Considering that synthesis of glycogen occurs by different pathways in mammals (via UDP-Glc) and bacteria (ADP-Glc), it is worth to highlight that ADP-Glc PPase can bevisualized as a particular target for controlling S. mutans virulence.