Structure and Enzymatic Properties of Genetically Truncated Forms of the Water-Insoluble Glucan-Synthesizing Glucosyltransferase from Streptococcus sobrinus

• Published in: Journal of biochemistry (Tokyo) 1999-08, Vol.126 (2), p.287-295
• Main Authors: Konishi, Norifumi, Torii, Yasuhiro, Yamamoto, Tatsuo, Miyagi, Atsushi, Ohta, Hiroyuki, Fukui, Kazuhiro, Hanamoto, Satoshi, Matsuno, Hideki, Komatsu, Hideyuki, Kodama, Takao, Katayama, Eisaku
• Format: Article
• Language: English
• Subjects: 3-glucan; Bacterial Proteins; C-terminal repeats; Catalysis; Cloning, Molecular; Codon; dextran; Dextrans - metabolism; domain structure; Dose-Response Relationship, Drug; Escherichia coli - enzymology; Gene Expression; glucans; Glucans - metabolism; glucosyltransfer-ase; Glucosyltransferases - metabolism; glycosyltransferase; Kinetics; Magnetic Resonance Spectroscopy; Microscopy, Electron; Models, Genetic; Peptide Fragments - metabolism; Protein Structure, Tertiary; Proteins - metabolism; Streptococcus - enzymology; Streptococcus sobrinus; Sucrose - pharmacology; Time Factors; Trypsin - pharmacology; α-l
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a022447

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (α-l, 6-glucan) into a water-insoluble form by making numerous α-l, 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-F 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 Km and kcat values at low concentrations (10 mM), however, GTF-I exhibited glucosyl transfer activity to form insoluble a-l, 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 α-l, 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.