Identifcation of reaction center of glycosyl transferase through molecular evolutionary and solid NMR analysis

通过分子进化和固体核磁共振分析鉴定糖基转移酶的反应中心

• 批准号: 15310155
• 负责人: SAITOU Naruya
• 金额: $ 10.24万
• 依托单位: National Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15310155/
• 关键词: glycosvl transferase; solid NMR; molecular evolution; E.coli; 3D structure of protein

We first conducted molecular evolutionary analyses of bacterial glycosyltransferase genes by using newly introduced PC cluster. We particularly focused on cluster of glycosyltransferase genes that define outer cell wall sugar structures that are know to vary from strain to strain of E. coli. Homology search was done by using these ca. 20 gycosyltransferase amino acid sequences as queries, and gene phylogenies were constructed for each homologous group. We found that one group of homologous genes changed their sugar specificities during evolution. This suggests that enzyme specificity was altered through a series of amino acid changes. We therefore chose waaO gene of E. coli K12 strain, and overexpressed its protein. The protein was purified with using His-tag affinity collum chromatography, and appropriate amount of purified protein was used for solid NMR analysis. Several clear native C13 peaks were observed using solid NMR method. We also added C13-labeled glucose to this purified protein, and found new C13 peak that may correspond to glucose-protein binding.

我们首次利用新引入的PC簇对细菌糖基转移酶基因进行了分子进化分析。我们特别关注定义外细胞壁糖基结构的糖基转移酶基因簇，已知这些糖基结构在不同菌株的大肠杆菌中是不同的。用这些约20个糖基转移酶氨基酸序列作为查询进行同源性搜索，并为每个同源组构建基因系统树。我们发现，一组同源基因在进化过程中改变了它们的糖特异性。这表明酶的特异性是通过一系列氨基酸的变化而改变的。因此，我们选择了大肠杆菌K12株的waaO基因，并对其蛋白进行了高效表达。用His-Tag亲和柱层析对该蛋白进行纯化，并用适量的纯化蛋白进行固体核磁共振分析。固体核磁共振法观察到几个清晰的天然C13峰。我们还在纯化的蛋白质中加入了C13标记的葡萄糖，并发现了可能对应于葡萄糖与蛋白质结合的新的C13峰。

项目成果

Katoh E.: "13C Nuclear Overhauser Polarization-Magic Angle Spinning (NOP-MAS) NMR Spectroscopy in uniformly 13C-labeled Solid Proteins"J.Am.Chem.Soc.. 126. 3653-3657 (2004)

Katoh E.：“均匀 13C 标记固体蛋白质中的 13C 核奥沃豪瑟偏振 - 魔角旋转 (NOP-MAS) NMR 光谱”J.Am.Chem.Soc.. 126. 3653-3657 (2004)

13C Nuclear Overhauser Polarization-Magic Angle Spinning (NOP-MAS) NMR Spectroscopy in uniformly 13C-labeled Solid Proteins.

统一 13C 标记固体蛋白中的 13C 核奥沃豪瑟偏振-魔角旋转 (NOP-MAS) NMR 光谱。

• 发表时间: 2004
• 期刊: J. Am. Chem. Soc. 126
• 作者: Katoh E.;Takegoshi K.;Terao T.
• 通讯作者: Terao T.

DNA sequence and comparative analysis of chimpanzee chromosome 22

• DOI: 10.1038/nature02564
• 发表时间: 2004-05-27
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Watanabe, H;Fujiyama, A;Sakaki, Y
• 通讯作者: Sakaki, Y

Takegoshi K.: "13C-1HDipolar-Driven 13C-13C Recoupling without 13C rf lrradiation in NMR of Rotating Solids"J.Chem.Phys.. 118. 2325-2341 (2003)

Takegoshi K.：“旋转固体 NMR 中无 13C rf 辐射的 13C-1HD 偶极驱动 13C-13C 重新耦合”J.Chem.Phys.. 118. 2325-2341 (2003)

Sequential Arrangement of γ-Valeolactone Enantiomers Enclathrated in Cholic Acids Channels as Studied by 13C Solid-State NMR : Elucidation of the Optical Resolution Mechanism

通过 13C 固态 NMR 研究包合在胆酸通道中的 γ-戊内酯对映体的顺序排列：阐明光学拆分机制

• 发表时间: 2004
• 期刊: J. Am. Chem. Socs. 126
• 作者: S.Nakamura