Genetic and functional diversity of β-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis

β- N -Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. β- N -Acetylgalactosaminidases hydrolyze β- N -acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-β- N -acetylgalactosaminidases that specifically act on β- N -acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight β- N -acetylgalactosaminidases and β- N -acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse β- N -acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity. Four β- N -acetylgalactosaminidase gene families were identified via deep-sea metagenome analysis. The biochemical and structural characterization of the aforementioned enzymes revealed their functional diversity and monophyletic evolutionary history.