A β-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.
Bibliographical noteFunding Information:
This study was supported by The Israel Science Foundation (Grant no. 676/00 to G.S. and Y.S.), and by The United States–Israel Binational Science Foundation (BSF) (Grant no. 96-178 to Y.S.), Jerusalem, Israel. Additional support was provided by the Fund for the Promotion of Research at the Technion, and by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany). V.B. acknowledges the financial support by the Center of Absorption in Science, the Ministry of Immigration Absorption and the Ministry of Science and Arts, Israel (Kamea Program).
- Acid-base catalyst
- Bacillus stearothermophilus
- Glycoside hydrolase family 39