The vast majority of glycosidases characterized to date follow one of the variations of the ‘Koshland’ mechanisms¹ to hydrolyse glycosidic bonds through substitution reactions. Here we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities². Using this, we identify a cluster of enzymes with extremely broad substrate specificities and thoroughly characterize these, mechanistically and structurally. These enzymes not only break glycosidic linkages of both α and β stereochemistry and multiple connectivities, but also cleave substrates that are not hydrolysed by standard glycosidases. These include thioglycosides, such as the glucosinolates from plants, and pseudoglycosidic bonds of pharmaceuticals such as acarbose. This is achieved through a distinct mechanism of hydrolysis that involves oxidation/reduction and elimination/hydration steps, each catalysed by enzyme modules that are in many cases interchangeable between organisms and substrate classes. Homologues of these enzymes occur in both Gram-positive and Gram-negative bacteria associated with the gut microbiome and other body parts, as well as other environments, such as soil and sea. Such alternative step-wise mechanisms appear to constitute largely unrecognized but abundant pathways for glycan degradation as part of the metabolism of carbohydrates in bacteria.

迄今为止，绝大多数糖苷酶都遵循“Koshland”机制 ¹ 的变体之一，通过取代反应水解糖苷键。在这里，我们描述了使用选择性鉴定非 Koshland 糖苷酶活性的测定法对人类肠道微生物组宏基因组库进行的大规模筛选 ² 。利用这一点，我们确定了一组具有极其广泛的底物特异性的酶，并从机制和结构上彻底表征了它们。这些酶不仅破坏 α 和 β 立体化学的糖苷键和多重连接性，而且还能裂解不被标准糖苷酶水解的底物。这些包括硫代糖苷，例如植物中的芥子油苷，以及药物的假糖苷键，例如阿卡波糖。这是通过一种独特的水解机制实现的，该机制涉及氧化/还原和消除/水合步骤，每个步骤都由酶模块催化，这些酶模块在许多情况下可以在生物体和底物类别之间互换。这些酶的同系物存在于与肠道微生物组和其他身体部位以及其他环境（例如土壤和海洋）相关的革兰氏阳性和革兰氏阴性细菌中。这种替代的逐步机制似乎构成了很大程度上未被识别但丰富的聚糖降解途径，作为细菌中碳水化合物代谢的一部分。