epiglucan and glucose-1-phosphate

1,3-β-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.

Topics: beta-Glucans; Disaccharides; Glucosephosphates; Hydrogen-Ion Concentration; Kinetics; Ochromonas; Phosphorylases; Proteoglycans; Substrate Specificity; Temperature

Cyclic beta-1,2-glucans (CbetaG) are osmolyte homopolysaccharides with a cyclic beta-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CbetaG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked beta-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a beta-1,2-glucan phosphorylase present at the CbetaG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the beta-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this "length-controlling" phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.

Topics: Amino Acid Sequence; Bacillus; beta-Glucans; Glucosephosphates; Glycosyltransferases; Molecular Sequence Data; Phosphorylases; Polysaccharides

2-Deoxy-D-glucose added to cultures of Candida albicans in the stationary phase of growth inhibited the incorporation of glucose into the (1----3)-beta-glucan fraction of the organisms. In the presence of ATP and cell extracts it was converted to 2-deoxy-D-glucose phosphate and when UTP was also present, material with the electrophoretic properties of UDP-2-deoxy-D-glucose was formed. In similar conditions glucose formed glucose phosphates, UDP-glucose and other products. Evidence was obtained that the analogue, after conversion to a phosphate derivative, was an inhibitor of phosphoglucomutase. When C. albicans was grown in the presence of 2-deoxy-D-glucose for 24 h, analogue residues became incorporated into the (1----3)-beta-glucan fraction and the subsequent rate of incorporation of glucose into that fraction was enhanced. The rate of turnover of glucose in this beta-glucan fraction was greater than in controls. Pretreatment of cultures with beta-glucanase, or incubation under conditions known to stimulate endogenous beta-glucanases, increased the subsequent rate of glucose incorporation and this increase was enhanced by growth in the presence of 2-deoxy-D-glucose. The analogue thus had the effect of altering the stability and glucose-acceptor function of (1----3)-beta-glucan chains. This could affect the properties of the polymer network leading to the known effect of the analogue in delaying the onset of phenotypic resistance to amphotericin methyl ester in stationary phase cultures of C. albicans.

Topics: Adenosine Triphosphate; beta-Glucans; beta-Glucosidase; Candida albicans; Deoxy Sugars; Deoxyglucose; Glucan 1,3-beta-Glucosidase; Glucans; Glucose; Glucosephosphates; Uridine Triphosphate