Department of Biotechnology, University of Tokyo, Japan.
FEBS J. 2014 Feb;281(3):778-86. doi: 10.1111/febs.12622. Epub 2013 Dec 12.
Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-α1,4-Glc), kojibiose (Glc-α1,2-Glc), trehalose (Glc-α1,α1,-Glc), and nigerose (Glc-α1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α-glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity.
糖苷水解酶（GH）家族 65 包含作用于麦芽糖（Glc-α1,4-Glc）、昆布二糖（Glc-α1,2-Glc）、海藻糖（Glc-α1,α1,-Glc）和黑曲霉二糖（Glc-α1,3-Glc）的磷酸化酶。这些磷酸化酶可以高效地催化具有高特异性的逆反应，因此可以应用于α-葡萄糖基低聚糖的实际合成。在这里，我们确定了来源于热纤梭菌的昆布二糖磷酸化酶与葡萄糖和磷酸盐以及与昆布二糖和硫酸盐复合物的晶体结构，首次提供了糖苷水解酶家族 65 酶对底物识别的结构见解。包含活性位点的昆布二糖磷酸化酶的环 3 区域明显长于其他酶的活性位点，并且围绕该环的三个残基，色氨酸 391、谷氨酸 392 和苏氨酸 417，识别昆布二糖。通过突变这三个残基，构建了各种模拟其他磷酸化酶残基保守模式的突变体。对这些突变体针对四种底物的活性测量表明，色氨酸 391 和谷氨酸 392，特别是后者，是昆布二糖活性所必需的。
