title: Stereochemistry of family 52 glycosyl hydrolases: a beta-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme.
abstract: 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 degrees C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xylopyranoside was followed by 1H-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 Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.
authors.affiliation: Department of Food Engineering and Biotechnology, Technion Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: T
authors.initials: T
authors.lastname: Bravman
authors.forename: G
authors.initials: G
authors.lastname: Zolotnitsky
authors.forename: S
authors.initials: S
authors.lastname: Shulami
authors.forename: V
authors.initials: V
authors.lastname: Belakhov
authors.forename: D
authors.initials: D
authors.lastname: Solomon
authors.forename: T
authors.initials: T
authors.lastname: Baasov
authors.forename: G
authors.initials: G
authors.lastname: Shoham
authors.forename: Y
authors.initials: Y
authors.lastname: Shoham
mesh_terms: D002384:Catalysis; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006027:Glycosides; C008646:4-nitrophenyl beta-D-xyloside; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase