Purification and characterization of a primer-independent glucosyltransferase from Streptococcus mutans 6715-13 mutant 27.
Affinity chromatography on Sephadex G-50 and subsequent ion-exchange chromatography on Trisacryl-M-DEAE were used to purify the glucosyltransferase (GTF) enzymes produced by mutant 27 of Streptococcus mutans 6715-13. Complete separation of three types of GTF, including a primer-independent GTF capable of synthesizing a slightly branched, water-soluble glucan (GTF-S), was obtained. The characteristics of this primer-independent GTF-S were compared with those of the normally occurring primer-dependent GTF-S. The Km for sucrose was easily obtained for each enzyme (10(-2) M), but the Km for dextran could only be determined for the primer-dependent GTF-S (5 X 10(-7) M for clinical dextran of molecular weight 60,000 to 90,000). The primer-independent GTF-S did not respond catalytically to the presence of either clinical dextran or the highly branched, water-soluble glucan produced by primer-dependent GTF-S, although it was capable of binding these polysaccharides at a noncatalytic site and of responding to the low-molecular-weight acceptor 1-O-methyl-alpha-D-glucopyranoside. The water-soluble glucan product of primer-independent GTF-S was a superior priming glucan for primer-dependent GTF enzymes as compared with the glucan product of primer-dependent GTF-S. The presence of primer-independent GTF-S in reaction mixtures stimulated glucan synthesis by primer-dependent GTF-S and by GTF synthesizing water-insoluble glucan by at least 10-fold, whereas the presence of similar amounts of primer-dependent GTF-S had no effect on synthesis by GTF synthesizing water-insoluble glucan. Primer-independent GTF-S appears to be a potent source of priming glucan for the primer-dependent GTF enzymes. Its possession of a noncatalytic binding site for glucan, the first observed for the GTF of S. mutans, suggests that it may also serve as a glucan receptor on the S. mutans cell surface.
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D002250:Carbon Radioisotopes; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005936:Glucans / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000302:isolation & purification*; D007700:Kinetics; D009154:Mutation; D013295:Streptococcus mutans / Q000201:enzymology*; D013395:Sucrose / Q000378:metabolism
D002250:Carbon Radioisotopes; D005936:Glucans; D013395:Sucrose; D005964:Glucosyltransferases