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

Galia Zaide, Dalia Shallom, Smadar Shulami, Gennady Zolotnitsky, Gali Golan, Timor Baasov, Gil Shoham, Yuval Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

Abstract

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 Km of 0.2 mM, and a specific activity of 42 U·mg-1 (kcat = 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.

Original languageAmerican English
Pages (from-to)3006-3016
Number of pages11
JournalEuropean Journal of Biochemistry
Volume268
Issue number10
DOIs
StatePublished - 2001

Keywords

  • Bacillus stearothermophilus
  • Family 67 glycosyl hydrolases
  • Inverting mechanism
  • Xylan
  • α-D-glucuronidase

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