Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.
The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs.
Shimamura
A
A
Department of Oral Biology, State University of New York, Buffalo 14214.
Nakano
Y J
YJ
Mukasa
H
H
Kuramitsu
H K
HK
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005798:Genes, Bacterial; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013295:Streptococcus mutans / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D005936:Glucans; D005964:Glucosyltransferases