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

Abstract 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 so far. In this study, we identified three novel glycoside hydrolase families distributed among all three domains of life and characterized eight novel 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 diversify their substrate specificities (endo-, dual-endo/exo-, and exo-) 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 novel evolutionary pathways for expanding their substrate specificity.