Structural and mechanistic insights into the cleavage of clustered O-glycan patches-containing glycoproteins by mucinases of the human gut

Mucinases of human gut bacteria cleave peptide bonds in mucins strictly depending on the presence of neighboring O-glycans. The Akkermansia muciniphila AM0627 mucinase cleaves specifically in between contiguous (bis) O-glycans of defined truncated structures, suggesting that this enzyme may recognize clustered O-glycan patches. Here, we report the structure and molecular mechanism of AM0627 in complex with a glycopeptide containing a bis-T (Gal beta 1-3GalNAc alpha 1-O-Ser/Thr) O-glycan, revealing that AM0627 recognizes both the sugar moieties and the peptide sequence. AM0627 exhibits preference for bis-T over bis-Tn (GalNAc alpha 1-O-Ser/Thr) O-glycopeptide substrates, with the first GalNAc residue being essential for cleavage. AM0627 follows a mechanism relying on a nucleophilic water molecule and a catalytic base Glu residue. Structural comparison among mucinases identifies a conserved Tyr engaged in sugar-pi interactions in both AM0627 and the Bacteroides thetaiotaomicron BT4244 mucinase as responsible for the common activity of these two mucinases with bis-T/Tn substrates. Our work illustrates how mucinases through tremendous flexibility adapt to the diversity in distribution and patterns of O-glycans on mucins. AM0627 is a bis-O-glycan mucinase that might work in the final steps of mucus degradation, thereby providing a carbon and nitrogen source for Akkermansia muciniphila. Here, the authors provide molecular insights into AM0627 function from X-ray crystallography and computer simulations.

Automated summary

This study provides molecular insights into the interaction between mucinase AM0627 and O-glycans in gut bacteria. The enzyme specifically cleaves bis-T O-glycopeptides by recognizing both the sugar moieties and the peptide sequence. Structural comparison reveals a conserved residue responsible for the common activity of AM0627 and another mucinase with bis-T/Tn substrates.