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Publication Abstract from PubMed

Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining alpha-glycoside hydrolases, such as alpha-glucosidase and alpha-xylosidase, among others. Recently, GH31 alpha-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 alpha-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 alpha-N-acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, (4)C1 --> (1)S3 --> (4)C1, in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes.

Structural and mechanistic insights into the substrate specificity and hydrolysis of GH31 alpha-N-acetylgalactosaminidase.,Miyazaki T, Ikegaya M, Alonso-Gil S Biochimie. 2021 Nov 23. pii: S0300-9084(21)00270-4. doi:, 10.1016/j.biochi.2021.11.007. PMID:34826537^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.