Stereochemistry of family 52 glycosyl hydrolases:: a β-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme

A beta -xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65 degreesC and pH 5.6-6.3, The stereochemistry of the hydrolysis of p-nitrophenyl beta -D-xylopyranoside was followed by H-1-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu313 are likely to be the two key catalytic residues involved in enzyme catalysis, (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.