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Analysis of the dextranase Gene of Cariogenic Bacteria

致龋菌葡聚糖酶基因分析

基本信息

批准号：
11671813
负责人：
IGARASHI Takeshi
金额：
$ 2.3万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671813/
关键词：
Dextranase Mutans streptococci Streptococcus mutans Cariogenic bacteria dex gene Active site Catalytic site う蝕原性細菌ミュータンスレンサ球菌 Streptococcus downei Streptococcus rattus

项目摘要

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.

本研究分析了龋菌的葡聚糖酶基因（dex基因）。首先测定了唐氏链球菌和rattus链球菌dex基因的核苷酸序列。S.downei dex基因的开放阅读框（ORF）全长3831 bp（1297个氨基酸残基），编码一个分子量为139743的葡聚糖酶（dextranase, dex）。该基因的ORF长度为2760 -bp（897个氨基酸残基），编码了一个长度为97596个氨基酸残基的dex。对产龋菌s.d down nei、s.d rattus、s.t mutans和s.d sobrinus葡聚糖酶的氨基酸序列进行比较，发现这些细菌的葡聚糖酶分子由两个可变区和一个保守区组成。其次，我们用分子遗传学方法研究了Dex催化位点的性质。通过比较葡萄糖基转移酶（Gtfs）的氨基酸序列，我们发现在葡萄糖基转移酶的保守区域和Gtfs的催化位点之间存在相似的序列（S.mutans Dex: 379-FDGWQGDTIGDN-390）。利用定点诱变技术将S.mutans Dex分子的Asp-385转化为Glu、Asn、Thr或Val。用任何氨基酸替换Asp-385导致Dex活性完全消失。另一方面，其他残基的替换不影响酶的活性。失活酶作为亲本酶仍具有右旋糖酐结合能力。这些结果表明，变形链球菌Dex中的Asp-385是维持酶活性所必需的，其他相关Dex中也存在类似的Asp残基。