Structural basis for N-glycopeptide preference in an infant-derived Bifidobacterium bifidum PNGaseA

N-glycans are abundant nutrient sources for human gut bacteria, which deploy peptide: N-glycosidases to release them from glycoproteins. Breast milk is comprised of a high proportion of N-glycosylated bioactive proteins that can be accessed by infant colonic microbes. Here, we biochemically and structurally characterize a PNGaseA-family enzyme from infant-associated Bifidobacterium bifidum LMG13195. This enzyme acts on a broad range of N-glycan structures but shows a marked preference for N-glycans attached to glycopeptides rather than to denatured or native proteins, highlighting a specialized route for N-glycan acquisition in infant-associated Bifidobacterium species. Structures of the apo enzyme, an unliganded E236Q mutant, and a D107N-E236Q complex with a G2-peptide reveal a canonical two-β-sandwich PNGase core plus an additional ten-strand β-sheet cradle and are the first structures of this superfamily. Comparison of these states, combined with molecular dynamics simulations, indicates that glycopeptide binding is coupled to β-sheet cradle-loop movements that close against β-sandwich 1 over the peptide C terminus. The complex defines recognition of the two core GlcNAc residues and the central mannose, reveals that peptide contacts are concentrated around the +2 Thr residue of the NXT sequon, and positions the scissile amide at the active site. These findings, together with mutational and computational analyses, provide a structural basis for N-glycopeptide preference by linking glycopeptide binding to cradle closure and catalysis, thereby expanding the host-glycan nutrient landscape accessible to infant-gut Bifidobacterium species.