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, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha-1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-glucan 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 characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure 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 simply hydrolyzed sucrose with similar K(m) and k(cat) values at low concentrations (<5 mM). At higher sucrose concentrations (>10 mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble alpha-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.
Konishi
N
N
Department of Operative Dentistry, Okayama University Dental School, Okayama, 700-8525, Japan.
Torii
Y
Y
Yamamoto
T
T
Miyagi
A
A
Ohta
H
H
Fukui
K
K
Hanamoto
S
S
Matsuno
H
H
Komatsu
H
H
Kodama
T
T
Katayama
E
E
D001426:Bacterial Proteins; D002384:Catalysis; D003001:Cloning, Molecular; D003062:Codon; D003911:Dextrans / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D008957:Models, Genetic; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D013291:Streptococcus / Q000201:enzymology*; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; D003062:Codon; D003911:Dextrans; D005936:Glucans; D010446:Peptide Fragments; D011506:Proteins; C067118:GTF-I protein, Streptococcus; D013395:Sucrose; D005964:Glucosyltransferases; C489364:glucosyltransferase I; D014357:Trypsin