Structural and mechanistic insights into the substrate specificity and hydrolysis of GH31 α-N-acetylgalactosaminidase.

Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining α-glycoside hydrolases, such as α-glucosidase and α-xylosidase, among others. Recently, GH31 α- N -acetylgalactosaminidases (Nag31s) have been identified to hydrolyze the core of mucin-type O -glycans and the crystal structure of a gut bacterium Enterococcus faecalis Nag31 has been reported. However, the mechanisms of substrate specificity and hydrolysis of Nag31s are not well investigated. Herein, we show that E. faecalis Nag31 has the ability to release N -acetylgalactosamine (GalNAc) from O -glycoproteins, such as fetuin and mucin, but has low activity against Tn antigen. Mutational analysis and crystal structures of the Michaelis complexes reveal that residues of the active site work in concert with their conformational changes to act on only α- N -acetylgalactosaminides. Docking simulations using GalNAc-attached peptides suggest that the enzyme mainly recognizes GalNAc and side chains of Ser/Thr, but not strictly other peptide residues. Moreover, quantum mechanics calculations indicate that the enzyme preferred p -nitrophenyl α- N -acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, 4 C 1 → 1 S 3 → 4 C 1 , in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes. [Display omitted] • EfGH31 released N -acetylgalactosamine from fetuin and bovine submaxillary mucin. • Crystal structures of EfGH31 complexed with substrates were determined. • The active site residues important for hydrolysis and substrate-binding were identified. • QM calculation revealed its conformational itinerary and reactivity. [ABSTRACT FROM AUTHOR]