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

α-d-Glucuronidases cleave the α-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 α-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 °C, 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 °C, pH 5.5–6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 °C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 °C. 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.