Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases
Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases
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对属于 GH5 糖苷酶新亚家族的新型神经酰胺内切酶 I 的广泛底物特异性的结构见解
DOI：
10.1074/jbc.m116.763821
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发表时间：
2017-03-24
影响因子：
4.8
通讯作者：
Yang, Guang-Yu
中科院分区：
文献类型：
--
作者：
Han, Yun-Bin;Chen, Liu-Qing;Yang, Guang-Yu
内切糖神经酰胺酶（EGCase）特异性水解各种鞘糖脂的寡糖和神经酰胺部分之间的糖苷键，在新兴的鞘糖脂学领域受到了广泛关注。然而，调节这些GH5糖苷酶严格底物特异性的机制尚未确定。在本研究中，我们报道了一种来自马红球菌103S的新型EGCase I（103S_EGCase I），其具有非常广泛的底物特异性。基于系统发育分析，该酶可能代表GH5糖苷酶的一个新亚家族。103S_EGCase I单独以及与底物单唾液酸二己糖基神经节苷脂（GM3）和单唾液酸四己糖基神经节苷脂（GM1）复合的X射线晶体结构使我们能够确定几个可能解释其广泛特异性的结构特征。与来自红球菌属M - 777的具有严格底物特异性的EGCase II（M777_EGCase II）相比，103S_EGCase I具有更长的α7 - 螺旋和更短的环4，这形成了一个更大的底物结合口袋，可以容纳更长的寡糖。此外，该酶的环2和环8采用更开放的构象，这也扩大了寡糖结合腔。基于此知识，进行了一项合理设计的实验来检测EGCase II的底物特异性。M777_EGCase II中环4的截短使其对GM1的活性提高了163%。值得注意的是，M777_EGCase II的S63G突变体显示出更广泛的底物谱，并且对大分子底物的活性显著提高（对岩藻糖基 - GM1高达>1370倍）。总之，这里呈现的结果揭示了EGCase精细的底物识别机制，并为这些酶的进一步改造提供了机会。
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 alpha 7-helix and a shorter loop 4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop 2 and loop 8 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 loop 4 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 recognitionmechanismofEGCasesandprovideanopportunityfor further engineering of these enzymes.