Analysis of the dextranase Gene of Cariogenic Bacteria

本研究对致龋菌的葡聚糖酶基因进行了分析。首先，测定了唐内链球菌和鼠链球菌dex基因的核苷酸序列。该基因的开放阅读框（ORF）为3，831-bp（1，297个氨基酸残基），编码葡聚糖酶（Dex），分子量为139，743。大鼠dex基因的ORF为2，760-bp（897个氨基酸残基），编码的Dex为97，596。通过比较致龋菌S.downei、S.rattus、S.mutans和S.sobrinus的Dex酶的氨基酸序列，发现这些致龋菌的Dex分子由两个可变区和一个保守区组成。通过比较葡萄糖基转移酶（Gtfs）的氨基酸序列，我们发现在Dexs的保守区和Gtfs的催化位点之间有一个相似的序列（S.mutans Dex：379-FDGWQGDTIGDN-390）。定点诱变用于将变形链球菌Dex分子的Asp-385转化为Glu、Asn、Thr或瓦尔。用任何氨基酸替换Asp-385导致Dex活性完全消失。另一方面，其他残基的取代不影响酶的活性。失活酶仍具有与亲本酶一样的葡聚糖结合能力。这些结果表明，变形链球菌Dex的Asp-385是酶活性所必需的，并且类似的Asp残基存在于其他相关Dex中。

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.