title: Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 β-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria.
abstract: The β-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues Asp(393), Glu(416) and Glu(364) was performed. NMR analysis of the xyloside hydrolysis products, the change of the reaction rate-limiting step for the Glu(416) mutants, the pH dependency of E416A activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining mechanism of glycoside hydrolysis, Glu(416) playing the role of acid/base catalyst and Asp(393) that of nucleophile.
authors: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors: Davide A
authors: DA
authors: Cecchini
authors: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors: Régis
authors: R
authors: Fauré
authors: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors: Elisabeth
authors: E
authors: Laville
authors: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France. Electronic address: veronese@insa-toulouse.fr.
authors: Gabrielle
authors: G
authors: Potocki-Veronese
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001644:Bifidobacterium / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D005965:Glucuronates / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007422:Intestines / Q000382:microbiology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005965:Glucuronates; D009844:Oligosaccharides; C570991:xylooligosaccharide; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: Bifidobacterium adolescentis; Catalytic residue; Glycoside hydrolase family 120; Retaining mechanism; Site-directed mutagenesis; β-Xylosidase