Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.
Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.
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细菌糖苷水解酶家族 63 与其糖合酶产物复合的结构，以及对底物特异性的深入了解。
DOI：
10.1111/febs.12424
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发表时间：
2013
期刊：
影响因子：
5.4
通讯作者：
Atsushi Nishikawa and Takashi Tonozuka
中科院分区：
文献类型：
--
作者：
Takatsugu Miyazaki;Megumi Ichikawa;Gaku Yokoi;Motomitsu Kitaoka;Haruhide Mori;Yoshikazu Kitano;Atsushi Nishikawa and Takashi Tonozuka
属于糖苷水解酶家族63（GH 63）的蛋白质存在于细菌、古细菌和真核生物中。虽然真核生物GH 63蛋白已被鉴定为加工α-葡萄糖苷酶I，但细菌和古细菌GH 63蛋白的底物特异性尚不清楚。在这里，我们将细菌GH 63酶，大肠杆菌YgjK，糖合酶，以探测其底物特异性。构建了YgjK的两个突变体（E727 A和D324 N），这两个突变体都显示出糖合酶活性。E727 A与β-d-葡萄糖基氟和单糖的反应表明，当半乳糖用作受体分子时，积累了最大量的糖合酶产物。与反应产物复合的E727 A的晶体结构表明，在活性位点结合的二糖是2-O-α-d-吡喃葡萄糖基-α-d-吡喃半乳糖（Glc 12 Gal）。通过比较E727 A-Glc 12 Gal和D324 N-蜜二糖的结构，发现它们的构象主要有两种类型：开放型和闭合型。当亚位点-1被葡萄糖占据时，YgjK的结构采用封闭形式。这些结果表明，含有Glc 12 Gal结构的糖是YgjK天然底物的最可能候选物。数据库E727 A-Glc 12 Gal和D324 N-melibiose的坐标和结构因子已分别以登录号3 W7 W和3 W7 X保藏在蛋白质数据库中
Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α‐glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme,Escherichia coliYgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with β‐d‐glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2‐O‐α‐d‐glucopyranosyl‐α‐d‐galactopyranose (Glc12Gal). A comparison of the structures of E727A–Glc12Gal and D324N–melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite −1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK.DatabaseThe coordinates and structure factors for E727A–Glc12Gal and D324N–melibiose have been deposited in the Protein Data Bank under accession numbers 3W7W and 3W7X, respectively