Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b)
Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b)
复制标题
使用保留 α-半乳糖苷酶的突变型多形拟杆菌 (BtGH97b) 高效合成 α-半乳糖基寡糖
DOI：
10.1111/febs.14018
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发表时间：
2016
期刊：
影响因子：
5.4
通讯作者：
Atsuo Kimura
中科院分区：
文献类型：
--
作者：
Masayuki Okuyama;Kana Matsunaga;Ken-ichi Watanabe;Keitaro Yamashita;Takayoshi Tagami;Asako Kikuchi;Min Ma;Patcharapa Klahan;Haruhide Mori;Min Yao;Atsuo Kimura
制备糖苷合酶（糖苷酶的催化亲核突变体）是有效合成糖苷键的公认策略。然而，衍生自α-糖苷酶的糖苷酶可产生所需产物的低产率，因为它们通常需要不稳定的β-糖基氟化物供体。在这里，我们研究了由来自糖苷水解酶家族（GH）97 α-半乳糖苷酶的催化亲核突变体催化的转糖基化，使用更稳定的β-半乳糖基叠氮化物和α-半乳糖基氟化物供体。该突变体酶在外部阴离子的辅助下使用β-半乳糖基叠氮化物和α-半乳糖基从α-半乳糖基氟化物转移来催化糖合酶反应。甲酸盐比叠氮化物更有效地恢复转移活性。动力学分析表明，在叠氮化物存在下转糖基化较差是因为酶、β-半乳糖基叠氮化物和受体之间的三元复合物活性低。解析了与转糖基化产物β-乳糖基α-d-半乳糖苷复合的突变酶的三维结构，以阐明α-半乳糖苷酶的配体结合方面。α-半乳糖苷酶的催化TIM桶的β→α环1、2和3与同源GH 97 α-葡萄糖苷水解酶的β→α环1、2和3的细微差异似乎涉及底物分解。特别地，β→α环1中的Trp残基具有不同的作用。α-半乳糖苷酶的Trp 312似乎排除了糖苷C4的赤道羟基，而α-葡萄糖苷水解酶中相应的Trp残基与该羟基形成氢键。α-半乳糖苷识别机制在GH 27、31、36和97 α-半乳糖苷酶中是保守的。数据库原子坐标（代码：5E 1 Q）已存入蛋白质数据库。
The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well‐established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from α‐glycosidases can give poor yields of desired products because they require generally unstable β‐glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α‐galactosidase, using more stable β‐galactosyl azide and α‐galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using β‐galactosyl azide and α‐galactosyl transfer from α‐galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, β‐galactosyl azide and acceptor. A three‐dimensional structure of the mutant enzyme in complex with the transglycosylation product, β‐lactosyl α‐d‐galactoside, was solved to elucidate the ligand‐binding aspects of the α‐galactosidase. Subtle differences at the β→α loops 1, 2 and 3 of the catalytic TIM barrel of the α‐galactosidase from those of a homologous GH97 α‐glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α loop 1 have separate roles. Trp312 of the α‐galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α‐glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α‐galactoside recognition is conserved among GH27, 31, 36 and 97 α‐galactosidases.DatabaseThe atomic coordinates (code: 5E1Q) have been deposited in the Protein Data Bank.