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Glycoside hydrolases of this family display alpha-glucuronidase activity. The enzymes target the glucuronic acid appended to the C2-OH of the xylose at the non-reducing end of xylooligosaccharides. The enzymes display a preference for 4-O-methyl-D-glucuronic acid side chains. The length of the oligosaccharide does not influence catalytic rate indicating that the enzyme only interacts with the uronic acid and the linked xylose. These enzymes do not remove glucuronic acid from internal regions of xylan [1, 2]. The enzymes are generally intracellular or membrane associated [3, 4] suggesting that they play a terminal role in uncapping decorated xyloooligosaccharides, making these molecules available to beta-xylosidases produced by the host.
Kinetics and Mechanism
α-glucuronidases are inverting enzymes that hydrolyse their target glycoside bond through a single displacement mechanism assisted by general acid and general base residues. Thus the glucuronic acid is formed in a beta configuration [5].
Catalytic Residues
Typical of inverting glycoside hydrolases, GH67 enzymes contain a general acid that protonates the scissile glycosidic oxygen promoting leaving group departure. This residue, Glu292 in the Cellvibrio japonicus GH67 [6] and Glu285 in the Geobacillus stearothermophilus GH67 enzymes [7] is a conserved glutamate within GH67. There are a pair of carboxylic acids that make hydrogen bonds with the catalytic water (attacks the anomeric carbon of the scissile glycosidic bond), and are predicted to activate the solvent molecule, thus acting as the general base. Which of these highly conserved residues, Glu393/Asp365 and Glu392/Asp364 in the C. japonicus and G. stearothermophilus enzymes, respectively, act as the general base is unclear. Mutational studies suggested that Asp365 in the C. japonicus enzyme may be the catalytic base [6], although similar mutagenesis studies on the Geobacillus glucuronidase indicate that mutation of either possible catalytic bases results in almost complete inactivation of the enzyme [8].
Three-dimensional structures
GH67 enzymes contain three distinct domains [6, 7]. The N-terminal domain forms a two-layer β sandwich, the central domain, the catalytic domain, is a classical (β/α)8 barrel whose catalytic center is located on the opposite, "C-terminal" side of the barrel to the N-terminal domain. The remaining, C-terminal domain is mainly α-helical. It wraps around the catalytic domain, making additional interactions both with the N-terminal domain of its parent monomer and also forming the majority of the dimer-surface with the equivalent C-terminal domain of the other monomer of the dimer. The active site comprises a deep, partially hydrophobic, pocket.
Family Firsts
First sterochemistry determination
1H NMR demonstrated that the released 4-methyl-D-glucuronic acid was a beta anomer and thus that the enzyme is an inverter [5].
