Molecular Analysis of Multiple Forms of the Streptococcus mutans Dextranase

采用分子遗传学方法对变形链球菌葡聚糖酶的多种形态进行了分子分析。为了找出蛀牙细菌葡聚糖酶（dextrnase, Dex）的共同特征，测定了唐氏葡萄球菌葡聚糖酶基因的全核苷酸序列，并与变形葡萄球菌和sobrinus进行了比较。同源性分析表明，Dex由一个保守区和两个可变区组成。此外，Dex活性染色检测到3种Dex均为活性多形态。因此，我们推测保守区对Dex活性至关重要，而N-末端和c -末端的可变区域负责Dex的多种形式。在此基础上，我们通过基因操作在突变链球菌Dex的两个可变区域构建突变体，并研究了对突变体多种形态和活性的影响。突变体在c端可变区表现出多种形式的Dex活性。突变体缺失c端可变区和部分保守区，不仅多种形式消失，而且活性也消失。另一方面，在具有Dex活性的c端突变体的n端可变区域内的突变体没有表现出多重形式和活性。这些结果表明，多种形式的Dex对c端可变区域的降解负责，并且表明保守区域可能包含活性位点和底物结合位点等催化位点。

Molecular analysis of multiple forms of the Streptococcus mutans dextranase was studied by the approach of molecular genetics. In order to find out common characteristics of dextrnase (Dex) of cariogenic bacteria, complete nucleotide sequence of the S. downei dextranase gene was determined and compared with those of S. mutans and S. sobrinus. Homology analysis showed that the Dex was constituted by a conserved region and two variable regions. In addition, Dex of three species was detected as active multiple forms in Dex active staining. We, therefore, speculated that the conserved region was essential for Dex activity and the variable regions of N- and C-terminals were responsible for the multiple forms of the Dex.. On the basis of this speculation, we constructed mutants at two variable regions on the S. mutans Dex by genetic manipulation and examined influence to the multiple forms and activity of the Dex. Mutants within a C-terminal variable region showed the multiple forms with the Dex activity. Mutants defective both the C-terminal variable region and a part of the conserved regions disappeared not only the multiple forms but also the activity. On the other hand, mutants within a N-terminal variable region of the C-terminal mutant with Dex activity did not show both the multiple forms and the activity. These results indicated that the multiple forms of the Dex was responsible for degradation of the variable region of C-terminus and suggested that the conserved region would contain the catalytic sites such as active and substrate binding sites.