Three-dimensional structural analysis and development of new inhibitors of glucan synthetase produced by cariogenic oral streptococci

远缘链球菌不溶性葡聚糖合成酶(GTF-I)是一种将蔗糖分解为葡萄糖和果糖，合成水不溶性葡聚糖的酶，属于口腔链球菌属。为了研究该酶的结构和功能，我们构建了GTF-I及其缺失突变体的高效表达系统，建立了高效的纯化方法，并对其结晶条件进行了优化。GTF-I由N端催化结构域(GS)和C端葡聚糖结合区(DXB)两个结构域组成。在结晶前，我们构建了完整的多肽GTF-I、N端缺失83个氨基酸的GS-6R和唯一的催化结构域GS三个多肽的大肠杆菌表达系统。采用硫酸铵沉淀法、离子交换层析法、亲和层析法和凝胶过滤层析法对每种多肽进行高效纯化。采用悬滴气相扩散法对每种多肽的最佳结晶条件(pH、盐浓度和沉淀剂的存在)进行了筛选。在完整的GTF-I中，在pH为6-8和盐浓度为0-0.2M的范围内都能获得微晶，但在所测试的任何条件下都没有观察到GS-6R蛋白的晶体形成。在GS中，在与GTF-I相似的条件下形成了许多微晶。此外，为了减少DXB结构域柔性的影响，在低温或在葡聚糖存在的情况下进行结晶。然而，晶体的质量并没有得到改善。虽然还没有得到适合于X射线结构分析的晶体，但进一步的筛选将有助于获得高质量的该酶晶体。

Insoluble glucan synthetase (GTF-I) of Streptococcus sobrinus, a member of oral streptococci, is an enzyme that splits sucrose into glucose and fructose and synthesizes water-insoluble glucan. For structure-function studies of this enzyme, we constructed overexpression systems of the GTF-I and its deletion mutants, established efficient purification methods, and investigated optimum conditions for crystallization. The GTF-I is composed two domains, N-terminal catalytic domain (GS) and C-terminal dextran-binding domain (DXB). Prior to crystallization, we constructed the Escherichia coli expression systems of three polypeptides, the complete peptide GTF-I, the N-terminal 83 amino acids-deleted GS-6R, and the only catalytic domain GS. Each polypeptide was highly purified with high efficiencies by combinations of ammonium sulfate precipitation, ion-exchange chromatography, affinity chromatography, and gel-filtration chromatography. Optimum conditions (pH, salt concentrations and the presence of precipitant agents) for crystallization were screened for each polypeptide using hanging-drop vapor diffusion method. In the complete GTF-I, micro-crystals were obtained in a pH range of pH 6-8 and in salt concentrations of 0-0.2 M. However, crystal formation of GS-6R protein was not observed in any conditions tested. In GS, many micro-crystals were formed in similar conditions to those in GTF-I. Furthermore, to reduce the effects of the flexibility of DXB domain, crystallization was performed at low temperature or at the presence of dextran. However, the quality of crystals was not improved. Although crystals suitable for X-ray structural analysis have not yet been obtained, further screening will lead to achieve high-quality crystals of this enzyme.