Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase)

Abstract

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound oa-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (-1,100 residues), that for glucan binding (-240 residues), and that of unknown function (-260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.Mutans streptococci produce several extracellular glucosyltransferases (GTFs) which synthesize water-soluble glucans and water-insoluble glucans (ISGs) containing co-1,6-and a-1,3-glucosyl linkages [ISG(1,6) and ISG(1,3), respectively]. The ISGs adhere to smooth tooth surfaces and facilitate aggregation of oral bacteria, so that GTFs are believed to play a key role in the formation of dental plaque (12,20).In view of this etiological importance, a number of studies of the properties of GTFs to understand the mechanism of ISG synthesis have been conducted over the past two decades (2,5,8,9,11,23,24,30,33). From the culture fluids of a strain of Streptococcus sobrinus (previously named Streptococcus mutans 6715), we purified an enzyme responsible for ISG synthesis (GTF-I) from sucrose in the presence of a-1,6 soluble glucan such as dextran T10 as a primer (9). In this reaction, sucrose is split into fructose and an enzymebound glucosyl moiety (26); the latter is transferred to the C-3 position of the glucose residue of the glucan, resulting in the formation of a-1,3-glucosyl polymer (GTF-I activity) (9). In the absence of the primer glucans, however, the glucosyl moiety is transferred to water (hydrolysis of sucrose or sucrase activity). Further studies have not been made because of a limited supply of purified GTF-I.In the meantime, the structure and function of GTFs have been studied by biochemical and recombinant DNA techniques. Glucan-binding fragments were isolated by tryptic digestion of GTF proteins from S. sobrinus, but none of them showed glucan-synthesizing activity (18,22). Of several molecular cloning studies of GTF genes from mutans streptococci (1,7,10,13,14,25...

Hypericin- and mTHPC-mediated photodynamic therapy for the treatment of cariogenic bacteria

“…The strongly different experimental sensitivity of the two streptococcus species observed in our study was therefore rather unexpected and question the close relationship. Our data support earlier molecular phylogenetic analyses of glucan-producing enzymes and superoxide dismutases indicating that the two bacteria are in fact genetically separate [35][36][37]. Currently, no comparable data of our mTHPC formulation are available for other microorganisms.…”

A circularly permuted α‐amylase‐type α/β‐barrel structure in glucan‐synthesizing glucosyltransferases

“…In fact sucrase activity is less affected by C-terminal deletion than glucosyltransferase, i.e. dextran synthesising, activity [48,50].…”

“…Thus in an L. mesenteroides dextran sucrase the sequence immediately preceding the predicted H3 has been implicated in sucrose binding and cleavage [12], while in glucosyltransferases the C-terminal region is recognised as a dextranbinding domain and a segment of ca. 300 residues following E3 is necessary for full transferase activity [10,[48][49][50]. It is, therefore, likely that the transferases utilise both regions equivalent to domain B in s-amylase, the N-terminal one being involved in sucrose scission and the C-terminal in glucosyl transfer to dextran.…”

Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

“…4,[6][7][8] The crystal structure of glucansucrases has not been determined, but from the alignments of their amino acid sequences, a permuted ð=Þ 8 barrel was deduced to be contained in their Nterminal catalytic domain. 9) A glucansucrase has been reported to hydrolyze sucrose in which all C-terminal repeating units are deleted, 10) but C-terminal repeat units are reported to be necessary for glucan-binding and glucan synthesis. 4,[6][7][8]10) Since the linkage patterns of glucans are different in various glucansucrases, funcy To whom correspondence should be addressed.…”

“…9) A glucansucrase has been reported to hydrolyze sucrose in which all C-terminal repeating units are deleted, 10) but C-terminal repeat units are reported to be necessary for glucan-binding and glucan synthesis. 4,[6][7][8]10) Since the linkage patterns of glucans are different in various glucansucrases, funcy To whom correspondence should be addressed. Fax: +81-29-838-8122; E-mail: funane@nfri.affrc.go.jp Abbreviations: DSRS, dextransucrase S; DSRT5, dextransucrase T5; dsrS, a gene that encodes DSRS; dsrT5, a gene that encodes DSRT5; GTF, glucosyltransferase; Glcp, glucopyranoside; SDS-PAGE, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis; Site 1, Tyr340-Asn510 of DSRS, Tyr307-Asn477 of DSRT5; Site 2, Lys696-Gly768 of DSRS, Lys668-Gly740 of DSRT5; Site 3, Asn917-Lys1131 of DSRS, Asn904-Lys1118 of DSRT5 tional amino acid positions or regions affecting the structure of the glucan product have been studied.…”