Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus

Abstract

A glucan-binding domain of 1,6-o-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.

Analysis of a dextran-binding domain of the dextranase of Streptococcus mutans

“…mutans Dex (Igarashi et al 1995), was necessary for dextran-binding ability. As glucan-binding proteins, such as Gtfs, Gbp and Dei, possess A and C repeats in glucanbinding sites (Ferretti et al 1987;Mooser and Wong 1988;Russell et al 1988;Banas et al 1990;Sun et al 1994), it was expected that the Dex, one of the glucan-binding proteins, would also have similar sequences. However, the expected sequences have not been detected in the Dex, implying that the binding mode of Dex to dextran is different from that of Gtfs, Gbp and Dei.…”

Glucansucrases: mechanism of action and structure–function relationships

“…Truncation of the C‐terminal end conducted to suppress glucan binding abilities of GTF‐I [21]. Mooser and Wong [88]as well as Kobayashi et al [89]have isolated by mild trypsic digestion of S. sobrinus GTF‐S and GTF‐I, peptides of 60.5 or 55 kDa, respectively, having the same affinity for glucan as native enzyme, but displaying no activity. Sequencing of peptides from GTF‐I of S. sobrinus showed that it corresponded to the C‐terminal part.…”

Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells

“…The decreased enzyme activity resulting from loss of direct repeats has been seen in other GTF enzymes (1,5,19) as well as in related pneumococcal enzymes with similar direct repeats (8). These decreased activities are thought to be due to decreased ability of the enzymes to bind their substrate (8,22). The results presented here show that the deletion of three internal direct repeats in strain CH107 did not decrease the amounts of cell-associated or extracellular GTF antibody-reactive protein, confirming that the decreased activities seen were due to a qualitative change in the enzyme.…”

“…gtfG encodes a series of six direct repeats in the carboxyl-terminal region. These conserved repeats, which in one classification system have been designated A, B, C, and D based on their similarity to consensus sequences (1,5,10,11), are thought to function in glucan binding (5,22), and are similar to ligand binding domains of proteins found in other gram-positive bacteria (34,36).…”