Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptoco ccus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha -1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-gluca n branches along the dextran chains with sucrose as the glucosyl donor, The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138 kDa) and GS (110 kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats char acteristic of streptococcal glucosyltransferases, GS lacks all of them, Ele ctron microscopy of negatively stained samples indicated a double-domain st ructure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I', In the absence of dextran, both fragments simp ly hydrolyzed sucrose with similar K-m and k(cat) values at low concentrati ons (<5 mM), At higher sucrose concentrations (>10 mM), however, GTF-I' exh ibited glucosyl transfer activity to form insoluble alpha-1,3-glucans. So d id GS, but less efficiently. Dextran increased the rate and efficiency of t he glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF -I' by mild trypsin treatment, this dextran-stimulated transfer was complet ely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are o rganized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-gl ucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as w ell as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.