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.