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Article Dans Une Revue Protein Engineering, Design and Selection Année : 2002

Probing the catalytically essential residues of the α-l-arabinofuranosidase from Thermobacillus xylanilyticus

Résumé

The α-l-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of α-l-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu28, Glu176 and Glu298 are important for catalytic activity. Kinetic data obtained for the mutant Glu176→Gln, combined with the results of chemical rescue using the mutant Glu176→Ala, have shown that Glu176 is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu176→Ala, indicated that α-l-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu298 might be the catalytic nucleophile and that Glu28 is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that α-l-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.

Dates et versions

hal-02074537 , version 1 (20-03-2019)

Identifiants

Citer

Takoua Debeche, Christophe C. Bliard, Philippe Debeire, Michael O'Donohue. Probing the catalytically essential residues of the α-l-arabinofuranosidase from Thermobacillus xylanilyticus. Protein Engineering, Design and Selection, 2002, 15 (1), pp.21-28. ⟨10.1093/protein/15.1.21⟩. ⟨hal-02074537⟩
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