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

Norifumi Konishi, Yasuhiro Torii, Tatsuo Yamamoto, Atsushi Miyagi, Hiroyuki Ohta, Kazuhiro Fukui, Satoshi Hanamoto, Hideki Matsuno, Hideyuki Komatsu, Takao Kodama, Eisaku Katayama

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (α-1,6-glucan) into a water-insoluble form by making numerous α-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 α-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 α-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.

Original languageEnglish
Pages (from-to)287-295
Number of pages9
JournalJournal of Biochemistry
Volume126
Issue number2
Publication statusPublished - 1999

Fingerprint

Streptococcus sobrinus
Glucosyltransferases
Glucans
Dextrans
Terminal Repeat Sequences
Water
Sucrose
Head
Trypsin
Electron microscopy
Hydrolysis
Electron Microscopy
Bacteria
Molecules

Keywords

  • α-1,3-glucan
  • C-terminal repeats
  • Dextran
  • Domain structure
  • Glucosyltransferase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus. / Konishi, Norifumi; Torii, Yasuhiro; Yamamoto, Tatsuo; Miyagi, Atsushi; Ohta, Hiroyuki; Fukui, Kazuhiro; Hanamoto, Satoshi; Matsuno, Hideki; Komatsu, Hideyuki; Kodama, Takao; Katayama, Eisaku.

In: Journal of Biochemistry, Vol. 126, No. 2, 1999, p. 287-295.

Research output: Contribution to journalArticle

Konishi, N, Torii, Y, Yamamoto, T, Miyagi, A, Ohta, H, Fukui, K, Hanamoto, S, Matsuno, H, Komatsu, H, Kodama, T & Katayama, E 1999, 'Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus', Journal of Biochemistry, vol. 126, no. 2, pp. 287-295.
Konishi, Norifumi ; Torii, Yasuhiro ; Yamamoto, Tatsuo ; Miyagi, Atsushi ; Ohta, Hiroyuki ; Fukui, Kazuhiro ; Hanamoto, Satoshi ; Matsuno, Hideki ; Komatsu, Hideyuki ; Kodama, Takao ; Katayama, Eisaku. / Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus. In: Journal of Biochemistry. 1999 ; Vol. 126, No. 2. pp. 287-295.
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AU - Torii, Yasuhiro

AU - Yamamoto, Tatsuo

AU - Miyagi, Atsushi

AU - Ohta, Hiroyuki

AU - Fukui, Kazuhiro

AU - Hanamoto, Satoshi

AU - Matsuno, Hideki

AU - Komatsu, Hideyuki

AU - Kodama, Takao

AU - Katayama, Eisaku

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N2 - Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (α-1,6-glucan) into a water-insoluble form by making numerous α-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 α-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 α-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.

AB - Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (α-1,6-glucan) into a water-insoluble form by making numerous α-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 α-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 α-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.

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