Structural dissection of the reaction mechanism of cellobiose phosphorylase
Structural dissection of the reaction mechanism of cellobiose phosphorylase
复制标题
DOI：
10.1042/bj20060274
复制
发表时间：
2006-08-15
影响因子：
4.1
通讯作者：
Fushinobu, Shinya
中科院分区：
文献类型：
--
作者：
Hidaka, Masafumi;Kitaoka, Motomitsu;Fushinobu, Shinya
纤维二糖磷酸化酶是糖苷水解酶家族94的一员，催化纤维二糖可逆磷酸解成α-D-葡萄糖1-磷酸和D-葡萄糖，同时转化异头构型。对纤维二糖磷酸化酶的底物特异性和反应机理进行了研究。我们已经确定了该酶的葡萄糖-硫酸盐和葡萄糖-磷酸盐复合物的晶体结构，最大分辨率为2.0埃（1埃= 0.1 nm）。磷酸根离子通过几个氢键被强烈地保持，并且该构型似乎适合于对异头中心的直接亲核攻击。糖供体和糖受体位点周围的结构特征与广泛的动力学研究的结果是一致的。当我们将此结构与同源壳二糖磷酸化酶的结构进行比较时，我们在糖供体和糖受体位点中确定了葡萄糖和N-乙酰葡萄糖胺之间底物区分的关键残基。我们发现纤维二糖磷酸化酶的活性位点口袋被一个额外的环所覆盖，这表明底物结合后需要一些构象变化。纤维二糖磷酸化酶的三维结构的信息将有助于这种酶的工程，其应用于实际的寡糖合成已经建立。
Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 angstrom (1 angstrom = 0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.