Knowledge‐based model of a glucosyltransferase from the oral bacterial group of mutans streptococci

Abstract

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of a-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with a-amylases and glucosyltransferase from Streptococcus downei gtfl (GTF). The significance of GTF similarity is underlined by GTF/a-amylase residues conserved in all but one a-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the a-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (P/a)8-barrel structure and belong to family 13 of the glycohydralases.Keywords: (P/a)x-barrel; catalytic residues; enzymes; mutans streptococci glucosyltransferase; protein modeling; site-directed mutagenesis Oral bacteria referred to as mutans streptococci secrete glucosyltransferases (EC2.4.1.5), which are a significant virulence factor in initiation of dental caries on smooth enamel surfaces and cementa1 root surfaces (Hamada & Slade, 1980;Loesche, 1986). The extracellular enzymes catalyze glucosyl transfer from sucrose to a growing glucan chain. The bacterial colony produces metabolic acids that demineralize the tooth surface and, if unchecked, will lead to dental caries.GTFs are very large proteins of approximately 1,300-1,700 residues. The enzyme can be grossly divided into an N-terminal catalytic domain and a C-terminal glucan-binding domain that captures the synthesized polysaccharide (Ferretti et al., Wong et al., 1990). The large size of the enzyme has made it difficult to develop structure information beyond the solution of the amino acid sequence. Nonetheless, we became aware of sequence similarity between GTF and members of the a-amylase family. In our earlier studies, we trapped a glucosyl-enzyme catalytic intermediate (Mooser & Iwaoka, 1989) that was coordinated with an aspartyl Reprint requests to: Gregory Mooser, Department of Basic Sciences, School of Dentistry, University of Southern California, Los Angeles, California 90089-0641; e-mail: gmooser@hsc.usc.edu.Abbreviations: GTF, glucosyltransferase from Streptococcus downei g@; MACAW, multiple alignment construction and analysis workbench; SP, sum of the pairs; dNJ, I-deoxynojirimycin. residue, and the sequence surrounding the aspartate was homologous with a sequence conserved in all members of the a-amylase family and several related enzymes (Mooser et al., Mooser, 1992).In this study, we coupled knowledge-based multiple sequence alignment with site-directed mutagenesis to establish evidence of structural similarity between GTF and solved crystal structures of members of the a-amylase family. The members of the GTF family are highly similar and ...

Mutans Streptococcal Infection Induces Salivary Antibody to Virulence Proteins and Associated Functional Domains

“…More than 50% of the infected animals showed a significant increase in responses reactive with the HDS peptide. This 19-mer peptide is associated with the beta 7 strand element of the putative (beta, alpha) 8 catalytic barrel domain of Gtf (8) and contains a catalytically essential histidine residue (His 461 in S. mutans GtfB) (42). Site-directed mutagenesis of a nearby aspartate (Asp 467 in S. mutans GtfB) within the HDS-associated Gtf sequence altered the water solubility of the glucan synthesized from sucrose (29).…”

“…Three monoepitopic peptide construct sequences (Pep 7, Pep 11, and Pep 16) were based on predicted MHC class II epitopes that were in the Gtf sequence, using the ProPred algorithm. Five monoepitopic peptide construct sequences (GGY, CAT, HDS, LVK, and VMAD) were based on Gtf sequences associated with putative catalytic interactions (8,9,(22)(23)(24)42). Two monoepitopic peptide construct sequences (GLB and GLU) were based on Gtf sequences associated with putative glucan binding domains in the C-terminal third of the Gtf protein (1,17,43).…”

“…In this regard one could follow development of salivary IgA antibody to glucosyltransferases (Gtf) and glucan binding protein B (GbpB), as well as intrinsic peptides with putative functional or major histocompatibility complex (MHC) binding roles, which have also been shown to elicit significant immune responses and, in some cases, protective immunity in experimental models. Such epitopes include several sequentially distinct residues in the N-terminal half of glucosyltransferase which are associated with its catalytic activity (8,9,16,(22)(23)(24)42). Also included would be natural immune responses to regions associated with the ability of Gtf to bind glucan through a series of repeating sequences in the C-terminal third of the molecule (1,17,43).…”

Novel Epitopic Region of Glucosyltransferase B from Streptococcus mutans

“…Secondary structure predictions suggest that the Gtfs are members of the ␣-amylase superfamily and contain a circularly permuted (␣/␤) 8 -barrel motif (8,19,26). Our results in Fig.…”

“…Since these observations suggested that the primary CAT site existx inside the (␣/␤) 8 -barrel structure, it was thought that this region may be difficult to recognize as an antigen for the immune system, although low-molecular-weight sucrose is able to pass through this structure as an enzyme substrate. Thus, it was suggested that the antigenicity of CAT would be low, as shown by Parker's prediction.…”