A β-Galactosidase acting on unique galactosides: the structure and function of a β-1,2-galactosidase fromBacteroides xylanisolvens, an intestinal bacterium

Abstract

Galactosides are major carbohydrates that are found in plant cell walls and various prebiotic oligosaccharides. Studying the detailed biochemical functions of beta-galactosidases in degrading these carbohydrates is important. In particular, identifying beta-galactosidases with new substrate specificities could help in the production of potentially beneficial oligosaccharides. In this study, we identified a beta-galactosidase with novel substrate specificity from Bacteroides xylanisolvens, an intestinal bacterium. The enzyme did not show hydrolytic activity toward natural beta;-galactosides during the first screening. However, when beta-D-galactosyl fluoride (beta-GalF) as a donor substrate and galactose or D-fucose as an acceptor substrate were incubated with a nucleophile mutant, reaction products were detected. The galactobiose produced from the beta-GalF and galactose was identified as beta-1,2-galactobiose using NMR. Kinetic analysis revealed that this enzyme effectively hydrolyzed beta-1,2-galactobiose and beta-1,2-galactotriose. In the complex structure with methyl beta-galactopyranose as a ligand, the ligand is only located at subsite +1. The 2-hydroxy group and the anomeric methyl group of methyl beta-galactopyranose faces in the direction of subsite +1 and the solvent, respectively. This observation is consistent with the substrate specificity of the enzyme regarding linkage position and chain length. Overall, we concluded that the enzyme is a beta-galactosidase acting on beta-1,2-galactooligosaccharides.