ABSTRACT
Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ß-1,3-galactan main chains and ß-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-ß-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ß-1,3-galactan, but did not act on ß-1,3-glucan, ß-1,3:1,4-glucan, xyloglucan, and agarose. It released various ß-1,3-galactooligosaccharides together with Gal from ß-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ß-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-ß-1,3-glucanase and endo-ß-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-ß-1,3-galactanase.