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

不溶性葡聚糖合成酶（Insoluble glucan synthetase, GTF-I）是一种将蔗糖分解为葡萄糖和果糖并合成不溶于水的葡聚糖的酶，是口腔链球菌的一员。为了研究该酶的结构-功能，我们构建了gtf - 1及其缺失突变体的过表达系统，建立了高效的纯化方法，并研究了最佳结晶条件。GTF-I由两个结构域组成，n端催化结构域（GS）和c端葡聚糖结合结构域（DXB）。在结晶之前，我们构建了三种多肽的大肠杆菌表达体系，即完整肽GTF-I、n端缺失83个氨基酸的GS- 6r和唯一催化结构域GS。通过硫酸铵沉淀、离子交换层析、亲和层析和凝胶过滤层析的组合，对每个多肽进行了高效纯化。采用悬垂气相扩散法筛选了每个多肽的最佳结晶条件（pH、盐浓度和沉淀剂的存在）。完整的gtf - 1在pH为6-8和盐浓度为0-0.2 m的条件下均可获得微晶，但在任何条件下均未观察到GS-6R蛋白的结晶形成。在GS中，许多微晶体的形成条件与GTF-I相似。此外，为了减少DXB结构域柔韧性的影响，在低温或葡聚糖存在下进行结晶。然而，晶体的质量并没有得到改善。虽然目前还没有获得适合x射线结构分析的晶体，但进一步的筛选将使该酶获得高质量的晶体。

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.