Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular aggregation.
The interaction between Streptococcus mutans dextransucrase (EC 2.4.1.5) and high-molecular-weight dextran was studied in both the presence and absence of substrate sucrose. Equivalent weight-percent solutions of primer dextrans that differed 200-fold in molecular weight were found to be equally efficient in priming new dextran synthesis. Sodium borohydride reduction of dextran had no effect on its priming ability. These results suggest that dextran synthesis proceeds by addition of glucosyl residues to nonreducing termini of primer dextrans and that several enzyme molecules simultaneously bind to single high-molecular-weight dextran molecules. Kinetic data suggested that dextransucrase contains only one dextran binding site per enzyme molecule. The nature of the commonly observed highly aggregated state of dextransucrase was also studied. Two types of enzyme aggregates were distinguished: (i) oligomeric enzyme aggregates that formed in the absence of dextran and were dissociated by 1 M KCl; and (ii) dextran-induced enzyme aggregates that were stable to 3 M salt. Oligomeric enzyme aggregates were obtained from supernatants of fructose-grown cultures, whereas dextran-induced enzyme aggregates appeared to be present in glucose-grown cultures. The molecular weight of the smallest species of dextran-free detransucrase observed in solutions of 1 M KCl was estimated to be 40,000 by gel column chromatography. Addition of dextran to primer-dependent dextransucrase resulted in formation of complexes that were stable in CsCl density gradients and exhibited a buoyant density of 1.382 g/cm3 as compared with a buoyant density of 1.302 g/cm3 exhibited by dextransucrase. The enzyme-dextran complexes observed in CsCl density gradients contained about 25% dextran. This corresponded to 150 enzyme molecules (molecular weight, 40,000) per dextran molecule (molecular weight, 2 X 10(6)). The implication of these results to the mechanism of sucrose- and dextran-induced aggregation of S. mutans is discussed.
D001665:Binding Sites; D002449:Cell Aggregation; D002499:Centrifugation, Density Gradient; D003911:Dextrans / Q000096:biosynthesis / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000032:analysis / Q000378:metabolism*; D066298:In Vitro Techniques; D008970:Molecular Weight; D013291:Streptococcus / Q000201:enzymology*; D013295:Streptococcus mutans / Q000201:enzymology*
D003911:Dextrans; D005632:Fructose; D005964:Glucosyltransferases; D005947:Glucose