Mechanism of Streptococcus mutans glucosyltransferases: hybrid-enzyme analysis

Abstract

Streptococcus mutans GS5 expresses three glucosyltransferases (GTFs): GTF-I and GTF-SI, which synthesize water-insoluble glucans in a primer-independent manner, and GTF-S, which is responsible for the formation of primer-dependent soluble glucan. The amino acid sequences of the GTF-I and GTF-S enzymes exhibit approximately 50% sequence identity. Various hybrid genes were constructed from the structural genes for the enzymes, and their products were analyzed. Three different approaches were used to construct the hybrid enzymes: (i) ligation of DNA fragments containing compatible endonuclease restriction sites of the two genes at homologous positions; (ii) in vivo recombination between the homologous regions of each gene; and (iii) random fusion of DNA fragments from each gene generated following exonuclease III digestion of tandemly arranged fragments corresponding to the two functional domains of each enzyme. Hybrid GTFs composed of the sucrose-binding domain of one enzyme (GTF-I or GTF-S) with the glucan-binding domain of the other synthesized insoluble glucan exclusively in the absence of primer dextran. Insoluble glucan synthesis by some, but not all, of the GTF-S:GTF-I chimeric enzymes was stimulated by primer dextran T10 addition. In addition, glucan binding by the former but not latter group of hybrid GTFs was demonstrated. These results suggest that the glucan-binding domain alone does not solely determine primer dependence or independence or the structure of the resulting glucan product, although this carboxyl-terminal domain containing direct repeating units does appear to play a significant role in primer dependence.

Analysis of functional domains of <i>Streptococcus sobrinus</i> glucosyltransferase U

“…Mooser et al (11) identified an active site of 9 amino acids in the N-terminal one-thirdof S. sobrinus GTFs , GTFI, Asp-Ser-Ile-Arg-ValAsp-Ala-Val-Asp, and GTFS, Asp-Gly-Val-Arg-Val-AspAla-Val-Asp. These consensus amino acid sequences are conserved in the GTFs from mutans streptococci and contain a putative catalyticAsp, which is common to a broad array and GtfD, showed that GBD was essential for glucan synthesis but not for sucrase activity (14). for GTFU are shown in Fig.…”

Three-dimensional modelling of the catalytic domain of Streptococcus mutans glucosyltransferase GtfB

“…This may allow the sucrose to enter the speci¢city pocket and to bind to the enzyme molecule at the active site. According to the results from previous studies which suggested that the C-terminal one-third is responsible for the glucan binding [9], we would expect the long molecule of glucan to occupy the remaining part or outside of this cavity and to be supported by the C-terminal residues. However, due to the elimination of residues for structural alignment, almost all the one-third C-terminal residues of the molecule cannot be seen in this structure.…”

“…(i) A C-terminal glucan-binding domain (GBD) is composed of multiple homologous direct repeat segments, approximately 510 residues [8]. GBD was shown to be essential for glucan synthesis but not for sucrase activity [9]. (ii) An N-terminal catalytic domain of about 900 amino acids [2] is capable of binding and the hydrolysis of sucrose [10].…”

Introduction of Raw Starch-Binding Domains into Bacillus subtilis α-Amylase by Fusion with the Starch-Binding Domain of Bacillus Cyclomaltodextrin Glucanotransferase

“…Some of these have been studied with regard to secretion of the enzyme (16), while others have been studied with regard to changes in substrate specificities (28,41), product specificities (29), or both (24). However, there have been few reports on hybrids of different enzyme species based on their domain-level organizations.…”