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-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 degrees C, were a Km of 0.2 mM, and a specific activity of 42 U x mg(-1) (kcat = 54.9 s(-1)). The enzyme was most active at 65 degrees C, pH 5.5-6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 degrees C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 degrees 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.
Zaide
G
G
Department of Food Engineering and Biotechnology, Technion - Israel Institute of Technology, Haifa, Israel.
Shallom
D
D
Shulami
S
S
Zolotnitsky
G
G
Golan
G
G
Baasov
T
T
Shoham
G
G
Shoham
Y
Y
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D020134:Catalytic Domain; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014306:Trioses / Q000737:chemistry
D014306:Trioses; D001224:Aspartic Acid; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; C096734:alpha-glucuronidase; D003545:Cysteine; D000409:Alanine