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

alpha -D-Glucuronidases cleave the alpha -1,2-glycosidic bond of the 4-O-me thyl-D-glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes. The alpha -D-glucuronidase from Bacillus stearothermop hilus T-6 was overexpressed in Escherichia coli using the T7 polymerase exp ression system. The purification procedure included two steps, heat treatme nt and gel filtration chromatography, and provided over 0.3 g of pure enzym e from 1 L of overnight culture. Based on gel filtration, the native protei n is comprised of two identical subunits. Kinetic constants with aldotetrao uronic acid as a substrate, at 55 degreesC, were a K-m of 0.2 mm, and a spe cific activity of 42 U.mg(-1) (k(cat) = 54.9 s(-1)). The enzyme was most ac tive at 65 degreesC, pH 5.5-6.0, in a 10-min assay, and retained 100% of it s activity following incubation at 70 degreesC for 20 min. Based on differe ntial scanning calorimetry, the protein denatured at 73.4 degreesC. Truncat ed 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, ind icating that the catalytic site is located in the central region of the pro tein. To identify the potential catalytic residues, site-directed mutagenes is 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 resid ues are the catalytic pair.