Analysis of domain interaction in insoluble glucan synthetase of oral streptococci

sobr inus链球菌的不溶性葡聚糖合成酶（GTF）是口腔链球菌的一员，由n端催化结构域（GS）和c端葡聚糖结合结构域（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由六个重复单元组成。我们构建了一系列GTF突变体，这些突变体具有GS与逐级删除重复单元（GS- 2r ~ GS- 6r）或具有GS和GBD结构域的反转（6R-GS），并用于本研究。结果如下：1. GBD是与右旋糖酐结合所必需的。GTF对葡聚糖的亲和力随着重复单位数的减少而降低。2. 右旋糖酐与GBD结合引起酶的结构变化。3. GBD中大缺失的GTF突变体虽然具有葡聚糖结合能力，但与小缺失的突变体相比，酶活性大大降低。4. 在GBD中有少量缺失的GTF突变中，两个结构域协同变性，表明两个结构域相互作用，而在GBD中有大量缺失的突变中，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的结合，还包括与GS的相互作用，从而实现两个结构域的功能耦合。这是该酶葡聚糖依赖性激活的原因。

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.