Characterization and interpretation of the reaction mechanism of a novel GH115 α-Glucuronidase from Flavobacteriaceae.

Xylan is a major component in the complex network of the plant cell wall. Xylan consists of xylose units connected by β-(1,4) glycosidic bonds, which can be heavily substituted, increasing its degradation recalcitrancy. Such substituents include α-D-glucuronic acid (glucopyranosyluronic acid, GlcAp) or 4-O-methyl GlcAp (MeGlcAp) at the O-2 position. Removal of these substituents can be catalyzed by α-glucuronidases. Here we report a novel two-domain α-glucuronidase from GH family 115, termed FAgu115A, that acts on both polymeric and oligomeric xylan. A homology model was constructed, revealing the significance of residues Asp303 and Asp177 for catalysis. These residues are conserved in all characterized proteins belonging to this family. The catalytic activity of these residues was verified by site-specific mutagenesis corroborating the hypothesis that Asp303 serves as the proton donor, while Asp177 increases the nucleophilicity of the reactive water through a hydrogen bond. Additionally, a potential CBM located at the C terminus is of importance for dimerization. Its significance was confirmed by producing a truncated variant followed by determination of the molecular mass by analytical size-exclusion chromatography. Kinetic and thermal inactivation analysis were performed resulting in a Km of 3.7 g/l on beechwood xylan and an activation energy (Eα) of 67 kJ/mol.