Regulation of bacterial glycogen synthesis
Besides glycogen, extracellular α-glucan and methyl glucose polysaccharide (MGLP) are polymers playing critical roles for Mtb physiology . Pathogenic mycobacteria are surrounded by a non-covalently bound capsule, whose major carbohydrate constituent is a glycogen like α-glucan. This cover plays a key role during the first stages of infection. Glycogen and α-glucan may even share in part a common biosynthetic route . Additionally, glucosyl-3-phosphoglycerate synthase (GpgS) from Mycobacterium bovis BCG catalyzes glucosylglycerate synthesis by condensation of NDP-Glc and 3-phosphoglycerate . This molecule is the precursor for the biosynthesis of MGLP participating in modulation of fatty acids elongation . GpgS utilizes both UDP-Glc and ADP-Glc, having a similar Vmax, although with a 6-fold higher affinity toward the former sugar nucleotide . The MGLP molecule is predicted to be elongated by the glycosyltransferase Rv3032 which also utilizes NDP-Glc, but the substrate specificity has yet to be reported in detail. Thus, the glucosyl transferase activity required for the glucan backbone of these three macromolecules (glycogen, α-glucan and MGLP) would be originally supplied with glucose building blocks coming from ADP-Glc PPase and/or UDP-Glc PPase characterized in this work. UDP-Glc PPase affinity for Glc-1P is 10-fold higher than ADP-Glc PPase affinity for same substrate (in absence of allosteric effector). In this context, Glc-1P would be constantly used to synthesize UDP-Glc, while its fluctuating consumption by ADP-Glc PPase would be mainly modulated toward the enzyme allosteric regulation by levels of Glc-6P and PEP ().
pyrophosphorylase by bromopyruvate at its allosteric activator site
The regulatory properties of the Mtb ADP-Glc PPase are distinct from those of other prokaryotes , but similar to the homologous protein from the related Gram-positive bacteria M. smegmatis and S. coelicolor. Despite some differences in the sensitivity to activation by Glc-6P and PEP, the fact that both ADP-Glc PPases from actinobacteria mainly respond to these effectors suggests the occurrence of similar domains involved in allosteric regulation given their close phylogenetic relatedness. The specificity toward effectors exhibited by the enzyme has commonalities with characteristics reported for the occurrence and modulation of other metabolic routes in mycobacteria. For example, M. smegmatis pyruvate kinase (catalyzing the conversion of PEP into pyruvate plus ATP) is activated by Glc-6P , and the hexose-P was reported as a key essential intermediate for mycobacterial metabolism .
Previous results indicated that glycogen content in retina fluctuated as the level of glucose changed in the incubation medium . Thus, in order to gain insight into the regulation of glycogen content in vivo, we studied glycogen levels and those of the GS- allosteric- activator G-6-P under different conditions.
with allosteric regulation of GP playing a ..
Tre-6P Sase. Synthesis of Tre-6P from NDP-Glc and Glc-6P was assayed by measuring NADH formation at 340 nm via the coupled spectrophotometric method previously utilized for other glycosyl transferases . The standard media contained 50 mM MOPS, pH 8.0, 5 mM MgCl2, 5 mM MnCl2, 0.3 mM phosphoenolpyruvate, 0.3 mM NADH, 2.5 mM NDP-Glc, 5 mM Glc-6P, 2 U pyruvate kinase, 2 U lactate dehydrogenase and 0.2 mg/ml BSA and appropriately diluted enzyme in a final volume of 100 μl. Reactions were incubated at 37 °C in a 96-well microplate and oxidation of NADH was followed at 340 nm using a Multiskan Ascent microplate reader (Thermo Electron Corporation). The conversion of substrates to the expected products was confirmed using proton NMR spectroscopy.
effectors when compared to allosteric systems of regulation
In general, efforts devoted to the characterization of enzymes related to glycogen metabolism in Gram-positive bacteria are scarce . Recently, our group approached this issue in Streptomyces coelicolor and Streptococcus mutans, where Glc-1P partitioning was understood to be controlled by the allosteric regulation of ADP-Glc PPase. In this work we extend this analysis to the metabolism in Mtb, studying the enzymes directing monosaccharides to glycogen and Tre synthesis. We report the molecular cloning and expression of Mtb genes coding for ADP-Glc PPase, UDP-Glc PPase, GSase and Tre-6P Sase and characterization of their recombinant products. Kinetic parameters were determined and ADP-Glc PPase regulatory properties were analyzed in detail. Biochemical data are discussed in the context of the metabolism of Tre, glycogen and α-glucan synthesis in mycobacteria, revealing how such a central metabolic node in the production of NDP-Glc is regulated in an important human pathogen.
allosteric regulation of GSK-3 by glycogen or ..
Besides, in spite of the significant changes observed in glycogen content under the diverse conditions studied, no differences in p-GS levels were found, which suggests that dephosporylation of GS was not an involved mechanism. Consistent with this interpretation, we found a significant correlation of G-6-P levels with GS activity as well as with glycogen content in retina (). Therefore, our results indicated that in retina G-6-P caused allosteric activation of GS, as has been reported in other tissues , , and that GS activation in turn led to the elevated glycogen content observed in diabetic retina. Thus, we conclude that retina glycogen content served as a glucose reservoir during periods of hypoglycemia and that its levels were regulated by G-6-P concentrations. The high levels of glycogen observed in the streptozotocin–treated rat retina were the result of the hyperglycemia, given that circulating glucose levels are the result of glucose release from liver and its removal from circulation by transport into muscle. The slow recovery of glycogen content in the retina from insulin-treated diabetic rats most likely represent a result of the chronic liver and muscle alterations of glucose disposal reported in type 1 diabetes , , . Therefore, since retina glycogen serves as an energy reservoir, under the hypoglycemia observed clinically in patients with type-1 diabetes, retinal neuronal function must be seriously compromised. Supporting this statement, glycemic control is a major concern in diabetic patients, particularly those having type 1, who undergo frequent hypoglycemic episodes . Under these conditions, the glycogen content in retina would be about 50% of the normal values and glucose production from glycogen would further support retinal function only for a short time through glycolytic metabolism. Indeed, rabbit and rat retinas incubated in the absence of glucose lead to decline in glycogen and ATP content, and loss of light-induced electrical signals within 30 min . Although it is clear that further studies will be needed to decipher the mechanisms, our results suggest that under the hypoglycemic conditions observed in diabetes, retinal neuronal survival could be compromised. In this respect, it is remarkable that decrease in retinal function was found in chronic hypoglycemia produced in an animal model . Moreover, during the revision of this manuscript, acute hypoglycemia was reported to induce retinal cells death in mouse and significant reduction of central retinal function in humans .