Probing the catalytically essential residues of the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus

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 Glu 2 8 , Glu 1 7 6 and Glu 2 9 8 are important for catalytic activity. Kinetic data obtained for the mutant Glu 1 7 6 →Gln, combined with the results of chemical rescue using the mutant Glu 1 7 6 →Ala, have shown that Glu 1 7 6 is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu 1 7 6 →Ala, indicated that a-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 Glu 2 9 8 might be the catalytic nucleophile and that Glu 2 8 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 a-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.