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Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis

Significance Host-adapted bacteria, both pathogenic and commensal, have developed multimodular enzymatic systems to cope with the complex glycans found in the host environment, such as the highly competitive gut niche. Among these deployed enzymatic arsenals are O-glycopeptidases, which uniquely target proteins with O-linked glycan modification. Through meticulous structural and functional analyses, we probe the molecular basis of glycan recognition and illuminate the complete structure of an ultramultimodular O-glycopeptidase. This provides extraordinary insight into glycan recognition in the catalytic site of this unique class of peptidase. It also reveals how noncatalytic carbohydrate binding by ancillary modules and substrate recognition in the O-glycopeptidase active site can be specifically coordinated in three-dimensional space. 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.