Functional conservation of divergent peptidase_M60 O-glycopeptidases in enterococcus.

O-glycopeptidases are proteolytic enzymes that obligately recognize the O-glycans appended to their substrates. Peptidase_M60 proteins comprise a superfamily of putative metal-dependent O-glycopeptidases that were initially described in host-associated bacteria but are now known to be distributed across bacteria occupying both host-associated and environmental niches. Although several members of this superfamily have been shown to possess O-glycopeptidase activity, the family is highly divergent at the amino acid sequence level, making it unclear whether this activity is conserved across all members. Here, we show that two peptidase_M60 enzymes, EfmM60 and EfcM60, from strains of Enterococcus faecium and Enterococcus faecalis, respectively, which are only distantly related at the primary sequence level to previously characterized O-glycopeptidases, exhibit both mucinase and O-glycopeptidase activity. Structural analysis of EfmM60 reveals distinct active-site features relative to previously characterized peptidase_M60 enzymes that provide a molecular basis for its ability to accommodate extended and branched O-glycans. Together, these findings highlight functional conservation within a highly divergent peptidase_M60 family and suggest that enterococcal O-glycopeptidases may contribute to ecological versatility by enabling access to O-glycosylated substrates across diverse biological contexts.