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Structural insights into the broad substrate specificity of a novel Endo-glycoceramidase I belonging to a new subfamily of GH5 glycosidase

  • [设施]:上海设施
  • [期刊/会议名称]:journal of biological chemistry
  • [摘要]:Endoglycoceramidases (EGCases) specifically hydrolyze the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids, and they have received substantial attention in the emerging field of glycosphingolipidology. However, the mechanism regulating the strict substrate specificity of these GH5 glycosidases has not been identified. In this study, we report a novel EGCase I from Rhodococcus equi 103S (103S_EGCase I) with remarkably broad substrate specificity. Based on phylogenetic analyses, the enzyme may represent a new subfamily of GH5 glycosidases. The X-ray crystal structures of 103S_EGCase I alone and in complex with its substrates monosialodihexosylganglioside (GM3) and monosialotetrahexosylganglioside (GM1) enabled us to identify several structural features that may account for its broad specificity. Compared with EGCase II from Rhodococcus sp. M-777 (M777_EGCase II), which possesses strict substrate specificity, 103S_EGCase I possesses a longer α7-helix and a shorter loop4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop2 and loop8 of the enzyme adopt a more open conformation, which also enlarges the oligosaccharide-binding cavity. Based on this knowledge, a rationally designed experiment was performed to examine the substrate specificity of EGCase II. The truncation of loop4 in M777_EGCase II increased its activity toward GM1 (163%). Remarkably, the S63G mutant of M777_EGCase II showed a broader substrate spectra and significantly increased activity toward bulky substrates (up to >1370-fold for fucosyl-GM1). Collectively, the results presented here reveal the exquisite substrate recognition mechanism of EGCases and provide an opportunity for further engineering of these enzymes.
  • [发表日期]:2017
  • [第一作者]:韩云宾,陈柳青
  • [第一作者单位]:上海交通大学
  • [通讯作者]:杨广宇
  • [通讯作者单位]:上海交通大学
  • [论文类型]:0
  • [期刊分类]:SCI2区
  • [学科分类]:生物学_生物化学
  • [影响因子]:4.258
  • [关键词]:Glycoside hydrolase, crystal structure, substrate specificity, protein engineering, sphingolipid
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