Identification of essential amino acids in the Streptococcus mutans glucosyltransferases

Abstract

to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp 411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp 491 or His 561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.The important role of the Streptococcus mutans glucosyltransferases (GTFs) (EC 2.4.1.5) in the induction of human dental caries has been well documented (16). These enzymes catalyze the synthesis of both water-insoluble glucans (IG) and soluble glucans (SG) from dietary sucrose. Human strains of S. mutans normally express three distinct GTFs, GTF-I and GTF-SI, which synthesize primarily ␣-1,3-rich glucans, and GTF-S, which produces ␣-1,6-linked glucans exclusively (1,7,8). The combined action of the three enzymes is required for maximum dental caries in experimental animals (32). Despite intensive investigation of these enzymes over the past decade by both molecular genetic (13) and biochemical (18) approaches, structure-function analysis of the GTFs has been initiated only recently (19,21,25). This is due mostly to the relatively large sizes of the enzymes (approximately 1,450 amino acids), which have made structural investigations difficult. Nevertheless, the isolation of the genes coding for these enzymes (13), as well as for related enzymes from other mutans streptococci (23) in addition to Leuconostoc mesenteroides (31), has made it possible to deduce structural similarities with other well studied enzymes, including ␣-amylases (17). These later approaches have suggested that the GTFs are members of the (␤/␣) 8 barrel-containing protein family. Since certain amino acid positions appear to be highly conserved in such proteins, it appeared to be possible to predict which amino acid residues of the GTFs are essential for activity. Recent results by both biochemical (20) and molecular genetic (10) approaches have identified the catalytic GTF Asp residue involved in covalent attachment of the glucose residue of sucrose to these enzymes. However, documentation of the roles of other amino acids in GTF catalysis has not yet been provided. Nevertheless, a number of amino acid positions in the S. mutans GTFs have been demonstrated to influence the nature of the glucan product synthesized (25). Based upon a comparison of the enzymes shown to be related to the GTFs, the present investigation utilized site-directed mutagenesis in order to establish the essential role ...

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

“…1F). Its mutation (His661Arg in L. mesenteroides NRRL B-512F DSRS and His561Gly in S. mutans GS-5 GTFB) resulted in very low residual enzyme activities (119,202). This residue thus may play a similar role in GS enzymes.…”

“…The seven conserved residues are indicated with arrows, and black arrows indicate the three catalytic residues. (A) Tyr residues, at positions 169 to 172 in GTFB of S. mutans GS5; mutation of these residues into Ala only changed the adhesiveness of the glucan products (202). (B) Thr344Leu, Glu349Leu, and His355Val of GTF-I, causing drops in enzyme activities of 30-, 4-, and 7-fold, respectively (122).…”

Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures

“…They are composed of two different functional domains (Monchois et al, 1999). The N-terminal catalytic domain (about 900 aa) is responsible for the cleavage of sucrose (Kato et al, 1992;Mooser et al, 1991;Tsumori et al, 1997). The C-terminal domain (300-400 aa) is composed of a series of homologous directly repeating units and is involved in glucan binding (Giffard & Jacques, 1994).…”

Dental Caries Vaccines: Prospects and Concerns

“…The catalytic activity of GTF appears to be associated with several, sequentially separate, residues in the N-terminal third of the molecule. These residues have been identified by a variety of methods, including the labeling of catalytic intermediates and site-directed mutagenesis (Mooser et al, 1991;Funane et al, 1993;Devulapalle et al, 1997;Tsumori et al, 1997;Monchois et al, 2000). Insight into catalytically important residue identification has been obtained by sequence alignment techniques (MacGregor et al, 1996;Devulapalle et al, 1997), which have revealed significant homology between GTFs and alpha amylase with respect to several invariant residues important to the catalytic activity of the alpha amylase family, suggesting that the amylase (␤,␣) 8 barrel element may also be a feature of the GTF catalytic domain.…”