Biochemical characterization and identification of catalytic residues in α-glucuronidase from Bacillus stearothermophilus T-6

alpha -D-Glucuronidases cleave the alpha -1,2-glycosidic bond of the 4-O-methyl-D-glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes. The alpha -D-glucuronidase from Bacillus stearothermophilus T-6 was overexpressed in Escherichia coli using the T7 polymerase expression system. The purification procedure included two steps, heat treatment and gel filtration chromatography, and provided over 0.3 g of pure enzyme from 1 L of overnight culture. Based on gel filtration, the native protein is comprised of two identical subunits. Kinetic constants with aldotetraouronic acid as a substrate, at 55 degreesC, were a K-m of 0.2 mm, and a specific activity of 42 U.mg(-1) (k(cat) = 54.9 s(-1)). The enzyme was most active at 65 degreesC, pH 5.5-6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 degreesC for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 degreesC. Truncated forms of the enzyme, lacking either 126 amino acids from its N-terminus or 81 amino acids from its C-terminus, exhibited low residual activity, indicating that the catalytic site is located in the central region of the protein. To identify the potential catalytic residues, site-directed mutagenesis was applied on highly conserved acidic amino acids in the central region. The replacements Glu392 --> Cys and Asp364 --> Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.