The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors

Abstract

The α--acetylgalactosaminidase from the probiotic bacterium (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAcα1-Ser/Thr). NagBb is involved in assimilation of-glycans on mucin glycoproteins by in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca, is involved in the recognition of the GalNAc -acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-α--acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.

Bifidobacterium dentium Fortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways

“…In contrast, the B. dentium Bd1 or DSM 20436 genomes do not encode any GH33 enzymes, indicating the inability to remove sialic acid. Some bifidobacteria also code for GH101 and GH129, which includes a cell wall-anchored endo-α- N -acetylgalactosaminidase that can remove entire glycan structures from the mucin protein (81, 94–98) (Fig. 2C and D).…”

Enzymatic Adaptation of Bifidobacterium bifidum to Host Glycans, Viewed from Glycoside Hydrolyases and Carbohydrate-Binding Modules

“…GNB and LNB are known to be imported by the GNB/LNB transporter, an ATP-binding cassette-type (ABC) transporter [75,76]. GH129 α-N-acetylgalactosaminidase (NagBb) is able to act on α-N-acetylgalactosaminyl Ser (Tn antigen) to liberate GalNAc and Ser [26,77]. GalNAc-Ser/Thr can be generated from core 3 or core 4 mucin O-glycans after complete trimming by concerted actions of GHs and peptidases.…”

Utilization of Host-Derived Glycans by Intestinal Lactobacillus and Bifidobacterium Species

“…This molecule is taken up by an as yet non-identified transporter and cleaved by the intracellular α- N -acetylgalactosaminidase NagBb that hydrolyzes the glycosidic bond between GalNAc and Ser/Thr amino acids ( Kiyohara et al, 2012 ). NagBb constitutes the first characterized member of the new GH129 family and the elucidation of its structure revealed the differences with GH101 enzymes regarding mechanism and substrate binding ( Sato et al, 2017 ).…”