Substrate recognition and cleavage by mucin degrading O-glycopeptidases from the gut microbe Bacteroides caccae.

Abstract

O-glycopeptidases are enzymes that hydrolyze the peptide bonds in glycoproteins by a mechanism that involves specific recognition of O-linked glycans on the substrate. Bacteroides caccae, an accomplished mucin degrader, is a member of the human gut microbiota with sixteen genes encoding putative O-glycopeptidases in the peptidase_M60 family. At present, the diversity of substrate selective in O- glycopeptidases is not well-understood nor is the rationale behind their expansion in bacteria such as B. caccae. Here we reveal the activity and diversity of the peptidase_M60 O-glycopeptidases encoded in the B. caccae genome. At least thirteen of the sixteen peptidase_M60 genes are active mucinases. Targeted functional studies by a high-throughput FRET screen combined with detailed kinetic analyses reveal that five examples in an uncharacterized clade of peptidase_M60 proteins have different substrate selectivities despite their relatively high degree of relatedness. Structural analyses of these enzymes, including bound complexes, reveal new insight into the molecular underpinnings of O-glycopeptidase diversity. This highlights the larger context of how varied the selectivity of peptidase_M60 O-glycopeptidases can be for the glycan moiety and/or the peptide portion of the substrates, and why mucin degraders like B. caccae diversify O-glycopeptidase substrate repertoires to potentially maximize breakdown of this extraordinarily complex polymer.