Identification of the Catalytic Nucleophile of the Family 29 α-l-Fucosidase from Sulfolobus solfataricus via Chemical Rescue of an Inactive Mutant

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Abstract
We have recently reported that a functional α-l-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622−14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-α-l-fucoside (pNp−Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the α-l-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 103-fold activity reduction on pNp−Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp−Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 α-l-fucosidases. This is the first time that this approach is used for α-l-glycosidases, widening the applicability of this method.

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