Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.

来自糖苷水解酶家族 43 (GH43) 的木聚糖分解酶参与半纤维素的分解，半纤维素是植物中第二丰富的碳水化合物。在这里，我们在动力学和机械方面描述了非还原端释放木糖的外切低聚木聚糖酶活性，并报告了天然 GH43 米氏复合物及其水解前底物的晶体结构。发现了活性位点木糖基单元的两种不同的钙稳定构象，表明有两种替代催化途径。这些结果得到了 QM/MM 模拟的证实，该模拟揭示了完整的水解机制并确定了两种可能的反应途径，涉及低聚木糖裂解的不同过渡态构象。糖苷酶中的这种催化构象混杂与活性位点的开放结构有关，因此可能扩展到其他外切酶。这些发现扩展了目前糖苷酶催化机制的一般模型，这是自然界中的一个主要反应，并影响了我们对它们与底物和抑制剂相互作用的理解。