A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately critical to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the molecular details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examination of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall molecular organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biological activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.

肽酶面临的一个挑战是识别高度多样化的底物。一些肽酶家族的一个特点是能够专门使用存在于其蛋白质底物上的翻译后添加的聚糖作为识别决定簇。这对于实现肽键水解至关重要。这类酶通常也很大且结构复杂。然而，支持这些O-糖肽酶识别聚糖的分子细节、这些相互作用的重要性及其辅助结构域的功能作用仍不清楚。在这里，使用产气荚膜梭菌ZmpA、ZmpB 和 ZmpC M60 肽酶作为模型蛋白，我们提供了结构和功能洞察，了解这些复杂的蛋白质如何将聚糖识别为催化和非催化底物识别的一部分。结构、动力学和诱变分析支持 M60 域催化位点内聚糖识别的关键作用，尽管它们指出 ZmpA 是一种明显无活性的酶。对 Zmp 结构域内容的更广泛检查揭示了非催化碳水化合物结合是这些蛋白质的一个特征。ZmpB 的完整三维结构提供了对高度多模块酶的整体分子组织的罕见洞察，并揭示了单个域功能的相互作用如何影响生物活性。哦-糖肽酶经常出现在寄居或攻击粘液层的适应宿主的微生物中。因此，我们预计这些结果将是提供更详细的模型的基础，以了解粘液中丰富的糖蛋白如何作为致病过程的一部分被破坏或在正常的共生生活方式中作为能量来源被释放。