Abstract
alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The substituents are removed by a repertoire of glycoside hydrolases and carbohydrate esterases including arabinofuranosidases, ␣-glucuronidases, and acetyl-xylan esterases, [2], whereas the xylose-containing polymeric backbone is hydrolyzed by endo-1,4-xylanases [2]
We show that the three positively charged residues play a critical role in substrate recognition and whereas Val-210 is not strictly required to accommodate the 4-O-methyl group of 4-O-MeGlcA, replacing this aliphatic amino acid with a polar residue greatly reduces the affinity of the enzyme for its substrate
Summary
Bacterial Strains, Plasmids, and Culture Conditions—The Escherichia coli strains used in this study were TUNER (DE3) obtained from Novagen and XL1 Blue. The Ki for the various reaction products was determined by measuring GlcA67A activity at four inhibitor concentrations between 0 and 200 mM, glucose; 0 and 200 mM, xylose; 0 and 50 mM, glucuronic acid (GlcA); 0 and 2 mM, 4-O-MeGlcA. The inhibitors were obtained from Sigma except 4-O-MeGlcA, which was prepared as follows: 800 mg of 4-O-methylglucuronoxylan (Sigma) was digested to completion with 800 units of the xylanase Xyn10A from C. japonicus [12] in 25 ml of 50 mM sodium phosphate, 12 mM citrate (PC) buffer, pH 6.5, for 16 h at 37 °C generating xylose, xylobiose, and aldotetraouronic acid as end products. Crystallization, Data Collection, and Processing—The E292A mutant of GlcA67A was crystallized as described previously [9] and soaked with aldobiouronic acid.
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