Analysis of the dextranase Gene of Cariogenic Bacteria

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In this study, we analyzed the dextranase gene (dex gene) of cariogenic bacteria. First of all, nucleotide sequence of the dex genes from Streptococcus downei and Streptococcus rattus were determined. Open reading frame (ORF) of the S.downei dex gene was 3,831-bp (1,297 amino acid residues) and coded a dextranase (Dex) with the molecular weight of 139,743. ORF of the S.rattus dex gene was 2,760-bp (897 amino acid residues) and encoded a Dex with that of 97,596. Comparison of amino acid sequences of the dextranases of cariogenic species, S.downei, S.rattus, S.mutans and S.sobrinus, showed that the Dex molecule of these bacteria consisted of two variable regions and a conserved region.Secondly, we examined the nature of the catalytic site of Dex by molecular genetic approaches. By comparing the amino acid sequences of glucosyltransferases (Gtfs), we noticed a similar sequence (S.mutans Dex : 379-FDGWQGDTIGDN-390) between the conserved region of Dexs and the catalytic site of Gtfs. Site-directed mutagenesis was used to convert the Asp-385 of the Dex molecule of S.mutans to either Glu, Asn, Thr, or Val. Replacement of Asp-385 with any of the amino acids resulted in complete disappearance of the Dex activities. On the other hand, replacement of other residues did not affect the enzyme activity. The inactive enzymes still possessed the dextran-binding ability as the parental enzyme. These results suggest that Asp-385 of the Dex of S.mutans was essential for enzyme activity and analogous Asp residues were present in other related Dexs.