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

Abstract

a-d-Glucuronidases cleave the a-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 a-dglucuronidase 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 8C, were a K m of 0.2 mm, and a specific activity of 42 U´mg 21 (k cat 54.9 s 21 ). The enzyme was most active at 65 8C, pH 5.5± 6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 8C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 8C. 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, sitedirected mutagenesis was applied on highly conserved acidic amino acids in the central region. The replacements Glu3923Cys and Asp3643Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.

The Membrane-Bound α-Glucuronidase from Pseudomonas cellulosa Hydrolyzes 4- O- Methyl- d -Glucuronoxylooligosaccharides but Not 4- O- Methyl- d -Glucuronoxylan

“…The polysaccharides are initially cleaved by xylanases into substituted xylooligosaccharides; enzymes associated with the bacterial surface then remove the side chains of these oligomers, and the liberated sugars, together with the xylooligosaccharides, are preferentially metabolized by the bacterium. We further suggest that the proposed role for P. cellulosa GlcA67A can be extended to other prokaryotic ␣-glucuronidases which have an intracellular location and appear to act on 4-O-methyl-D-glucuronoxylooligosaccharides but not on 4-O-methyl-D-glucuronoxylan (21,26).…”

Purification and Characterization of a Novel α-Glucuronidase fromAspergillus nigerSpecific forO-α-D-Glucosyluronic Acid α-D-Glucosiduronic Acid

“…Most of them have been ranked as hydrolase, which degrade side chains of xylan containing -1,2 glucuronyl linkages. [1][2][3][4][5][6][7][8][9] For instance, intracellular -glucuronidase from A. niger easily hydrolyzed Me-GA-X n ( Table 3).…”

“…1,2,[6][7][8] It is known that -glucuronidases from bacteria and snail are composed of two subunits (Table 2). [3][4][5]9,11) Effects of pH, temperature, and chemicals As shown in Fig. 2, the enzyme was stable in a pH range of 2.0-4.0 at 30 C, and the optimum pH was pH 3.0-3.5.…”

“…TreDCase had the most acidic optimum pH among the fungal and bacterial -glucuronidases previously reported (Table 2). [2][3][4][5][6][7][8][9] The enzyme was stable up to 30 C after incubation for 24 h at pH 4.0, and activity was highest at 40 C. When the enzyme was treated with 2.0 mM of various chemicals and metal ions, Hg 2þ (residual activity, 42%) and SDS (52%) inhibited activity (data not shown). K þ , Na þ , Ca 2þ , Cu 2þ , Mg 2þ , Ag 2þ , and dithiothreitol slightly activated TreDCase (110-118%), and Mn 2þ , Fe 3þ , and Fe 2þ marginally inhibited it (80-87%).…”

“…[1][2][3][4][5][6][7][8][9] Most microbialglucuronidases degrade -1,2 glucuronyl linkages to liberate 4-O-methyl D-glucuronic acids, which exist as side chains of xylan, although the enzymes do not hydrolyze p-nitrophenyl-O--D-glucosiduronic acid (PNP -glucuronide). [1][2][3][4][5][6][7][8] Among microbial -glucuronidases, -glucuronidase from Thermotoga maritima is the only enzyme that was known to hydrolyze PNPglucuronide. 9) Substrate specificities of the -glucuronidases, however, have not been examined in detail, due to the unavailability of various -glucuronyl oligosaccharides.…”