title: Identification of essential amino acids in the Streptococcus mutans glucosyltransferases.
abstract: A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 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 Trp491 or His561 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.
authors.affiliation: Department of Oral Biology, State University of New York, Buffalo 14214, USA.
authors.forename: H
authors.initials: H
authors.lastname: Tsumori
authors.forename: T
authors.initials: T
authors.lastname: Minami
authors.forename: H K
authors.initials: HK
authors.lastname: Kuramitsu
mesh_terms: D000595:Amino Acid Sequence; D000601:Amino Acids, Essential / Q000235:genetics*; D001483:Base Sequence; D017124:Conserved Sequence; D005964:Glucosyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*
chemical_list: D000601:Amino Acids, Essential; D005964:Glucosyltransferases