期刊
BIOCHIMIE
卷 195, 期 -, 页码 90-99出版社
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2021.11.007
关键词
Gut bacteria; Mucin; N-acetylgalactosamine; O-glycan; Quantum mechanics
资金
- Japan Society for the Promotion of Science KAKENHI [19K15748]
- European Union [847548]
- Marie Curie Actions (MSCA) [847548] Funding Source: Marie Curie Actions (MSCA)
Glycoside hydrolase family 31 (GH31) is a diverse family of anomer-retaining alpha-glycoside hydrolases. Recently, GH31 alpha-N-acetylgalactosaminidases (Nag31s) have been found to hydrolyze mucin-type O-glycans and the crystal structure of Enterococcus faecalis Nag31 has been reported. This study investigates the substrate specificity and hydrolysis mechanism of E. faecalis Nag31, revealing its ability to release N-acetylgalactosamine from O-glycoproteins and providing insights into the diversification of sugar recognition and hydrolysis mechanisms in GH31 enzymes.
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, C-4(1) -> S-1(3) -> C-4(1), in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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