IDENTIFICATION OF ESSENTIAL AMINO-ACIDS IN THE STREPTOCOCCUS-MUTANS GLUCOSYLTRANSFERASES

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 h ave been implicated as essential in catalysis. Utilizing a site-direct ed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp (413), Trp(491), and His(561), as being important in enzymatic activit y. Mutagenesis of Asp(413) to, Thr resulted in a GTF which expressed o nly about 12% of the wild-type activity. In contrast, mutagenesis of A sp(411) did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escher ichia coli. Moreover, conversion of Trp(491) Or His(561) to either Gly or Ata resulted in enzymes devoid of GTF activity, indicating the ess ential nature of these two amino acids for activity. Furthermore, muta genesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequenc es present in the glucan-binding domain of the GTFS had little overall effect on enzymatic activity, although the glucan products appeared t o be less adhesive. These results are discussed relative to the mechan isms of catalysis proposed for the GTFs and related enzymes.