Crystal structure of the Enterococcus faecalis α-N-acetylgalactosaminidase, a member of the glycoside hydrolase family 31.

Glycoside hydrolases catalyze the hydrolysis of glycosidic linkages in carbohydrates. The glycoside hydrolase family 31 (GH31) contains α-glucosidase, α-xylosidase, α-galactosidase, and α-transglycosylase. Recent work has expanded the diversity of substrate specificity of GH31 enzymes, and α-N-acetylgalactosaminidases (αGalNAcases) belonging to GH31 have been identified in human gut bacteria. Here, we determined the first crystal structure of a truncated form of GH31 αGalNAcase from the human gut bacterium Enterococcus faecalis. The enzyme has a similar fold to other reported GH31 enzymes and an additional fibronectin type 3-like domain. Additionally, the structure in complex with N-acetylgalactosamine reveals that conformations of the active site residues, including its catalytic nucleophile, change to recognize the ligand. Our structural analysis provides insight into the substrate recognition and catalytic mechanism of GH31 αGalNAcases.

Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae.

Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2(1) and diffracted to 2.0 A resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 A, beta = 112.46 degrees. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-alpha-D-galactopyranoside, which is consistent with the classification of this enzyme as an endo-alpha-N-acetylgalactosaminidase.