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

Abstract

Abstractβ-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.

Bioinformatic, Enzymatic, and Structural Characterization of Trichuris suis Hexosaminidase HEX-2

“…The presence of a Zn 2+ ion near the proposed active site was not expected, its role is unknown but this is found in some other glycosidases, including Golgi mannosidase II. 48 Another example monosaccharide-releasing hexosaminidase with a high GalNAc-bias is the recently crystallized Paenobacillus TS12 NgaP2 (PDB 8K2L); 49 despite overall structural superposition of their catalytic regions being possible, a meaningful comparison regarding side chains determining substrate specificity is difficult as NgaP2, assigned to the GH123 family, has only 11% identity with HEX-2. Few comparisons can be made to eukaryotic GH20 hexosaminidases as the four proteins for which there are crystallographic data are all of subfamily 2.…”