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Abstract：
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.
摘要：
糖苷水解酶（GH）家族65包含作用于麦芽糖的磷酸化酶（Glc-α1,4-Glc），曲二糖（Glc-α1,2-Glc），海藻糖（Glc-α1，α1，-Glc），和nigerose(Glc-α1,3-Glc)。这些磷酸化酶可以高效催化具有高特异性的逆反应，因此可应用于α-葡萄糖基寡糖的实际合成。这里,我们确定了与葡萄糖和磷酸盐复合以及与曲比糖和硫酸盐复合的Caldicellulosilicolyticus的曲二糖磷酸化酶的晶体结构，提供了对糖苷水解酶家族65酶的底物识别的第一个结构见解。包含曲二糖磷酸化酶活性位点的第3环区域明显长于其他酶的活性位点，和围绕这个环的三个残基，Trp391、Glu392和Thr417可识别曲比糖。通过在这三个残基处的突变来构建模拟其他磷酸化酶的残基保守模式的各种突变体。突变体对四种底物的活性测量表明Trp391和Glu392，尤其是后者，是曲别糖活动所必需的。
