Analysis of domain interaction in insoluble glucan synthetase of oral streptococci

嗜水链球菌不溶性葡聚糖合成酶(GTF)是口腔链球菌的一员，它由N端的催化域(GS)和C端的葡聚糖结合域(GBD)组成。在这项研究中，我们研究了这两个结构域之间的相互作用，以及它对酶活性的影响。GBD由六个重复单位组成。我们构建了一系列GTF突变体，这些突变体具有GS和逐步删除的重复单位(GS-2R~GS-6R)或具有GS和GBD结构域的倒置(6R-GS)，并用于本研究。研究结果如下。1.结合葡聚糖离不开GBD。GTF对葡聚糖的亲和力随着重复单位数的减少而降低。2.葡聚糖与GBD的结合引起了酶的结构变化。3.GBD基因缺失较大的GTF突变体虽然具有与葡聚糖结合的能力，但与缺失较少的突变体相比，GTF的酶活性显著降低。4.在GBD基因缺失较少的GTF突变株中，两个结构域协同变性，提示两个结构域的相互作用，而GBD基因缺失较大的突变株，两个结构域独立变性。综上所述，GBD的作用不仅包括与葡聚糖结合，还包括与GS相互作用，从而实现两个结构域的功能偶联。这是依赖葡聚糖激活这种酶的原因。

Insoluble glucan synthetase (GTF) of Streptococcus sobr inus, a member of oral streptococci, is composed of a catalytic domain (GS) in the N-terminal side and a dextran-binding domain (GBD) in the C-terminal side. In this study, we examined the interaction between the two domains, and its effects on the enzymatic activity. GBD consists of six repeat units. We constructed a series of the GTF mutants that have GS with step-wise deleted repeat units (GS-2R 〜 GS-6R) or have an inversion of GS and GBD domains (6R-GS), and used in this study. The results are as follows. 1. GBD was indispensable for binding to dextran. The affinity of GTF for dextran was decreased with decrease of the number of repeat units. 2. Binding of dextran to GBD caused structural change in the enzyme. 3. The GTF mutants with a large deletion in GBD, although having a dextran-binding ability, showed greatly decreased enzymatic activity compared to mutants with a small deletion. 4. In GTF mutatns with a small deletion in GBD, two domains denatured cooperatively, suggesting the interaction of two domains, whereas mutants with a large deletion in GBD, two domains denatured independently. Taken together, the roles of GBD include not only binding to dextran, but also interaction with GS, which enables functional coupling of two domains. This is responsible for dextran-dependent activation of this enzyme.