An essential amino acid residue for catalytic activity of the dextranase of Streptococcus mutans.
Dextranase (Dex) is an enzyme that hydrolyzes glucan, a polymer of glucose synthesized from sucrose by glucosyltransferases (GTFs). By comparing amino acid sequences of Dexs and GTFs, we found that the Dex enzymes of Streptococcus mutans, Streptococcus sobrinus, Streptococcus downei and Streptococcus salivarius had similar amino acid sequences to those of the catalytic sites of GTFs of mutans streptococci. We therefore examined the amino acid essential in Dex catalysis by molecular genetic approaches in this study. Site-directed mutagenesis was used to convert the Asp-385 of the Dex molecule of S. mutans Ingbritt to Glu, Asn, Thr or Val. Replacement of Asp-385 with any of the amino acids resulted in complete disappearance of Dex activity. However, replacement of other Asp residues did not affect the enzyme activity. The inactive enzymes still retained dextran-binding ability. These results suggest that Asp-385 of the Dex of S. mutans Ingbritt was essential for enzyme activity and the catalytic and substrate-binding sites were located at different sites within the Dex molecule.
Igarashi
T
T
Department of Oral Microbiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
Morisaki
H
H
Yamamoto
A
A
Goto
N
N
D000595:Amino Acid Sequence; D000601:Amino Acids, Essential / Q000235:genetics*; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003910:Dextranase / Q000235:genetics*; D005964:Glucosyltransferases / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008967:Molecular Biology; D016297:Mutagenesis, Site-Directed; D020539:Sequence Analysis, Protein; D013291:Streptococcus / Q000145:classification / Q000201:enzymology; D013295:Streptococcus mutans / Q000201:enzymology*; D016986:Streptococcus sobrinus / Q000201:enzymology; D013912:Threonine / Q000235:genetics; D014633:Valine / Q000235:genetics
D000601:Amino Acids, Essential; D013912:Threonine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D005964:Glucosyltransferases; D003910:Dextranase; D014633:Valine