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

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