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

致龋口腔链球菌新型葡聚糖合成酶抑制剂的三维结构分析及开发

• 批准号: 13557161
• 负责人: FUKUI Kazuhiro
• 金额: $ 7.55万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557161/
• 关键词: insoluble glucan synthease; oral streptococci; dextran; secondary structure; GTF-I; Streptococcus; catalytic domain; crystallization; グルカン合成酵素; 口腔レンサ球菌; 立体構造解析; GTF; X線結晶解析; 大量発現系; 立体構造; 不溶性グルカン合成酵素

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.

远缘链球菌（Streptococcus sobrinus）的不溶性葡聚糖合成酶（GTF-1）是一种将蔗糖分解为葡萄糖和果糖并合成水不溶性葡聚糖的酶。为了研究该酶的结构与功能，我们构建了GTF-1及其缺失突变体的过表达系统，建立了有效的纯化方法，并研究了结晶的最佳条件。GTF-1由N端催化结构域（GS）和C端葡聚糖结合结构域（DXB）组成。在结晶之前，我们构建了三种多肽的大肠杆菌表达系统，即完整的肽GTF-I、N-末端83个氨基酸缺失的GS-6 R和唯一的催化结构域GS。通过硫酸铵沉淀、离子交换色谱、亲和色谱和凝胶过滤色谱的组合，以高效率高度纯化每种多肽。使用悬滴气相扩散法筛选每种多肽的最佳结晶条件（pH、盐浓度和沉淀剂的存在）。在完整的GTF-1中，在pH 6-8的pH范围和0- 0.2M的盐浓度下获得微晶。然而，在任何测试条件下均未观察到GS-6 R蛋白的晶体形成。在GS中，许多微晶在与GTF-1中的那些类似的条件下形成。此外，为了减少DXB结构域的柔性的影响，在低温下或在葡聚糖的存在下进行结晶。然而，晶体的质量并没有得到改善。虽然尚未获得适合于X射线结构分析的晶体，但进一步筛选将导致获得该酶的高质量晶体。