| Abstract
| - We have recently reported that a functional α-l-fucosidase could be expressed by a singleinsertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of thehyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278,14622−14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolarspecificity for p-nitrophenyl-α-l-fucoside (pNp−Fuc) and promoted transfucosylation reactions by followinga reaction mechanism in which the products retained the anomeric configuration of the substrate. Theactive site residues in GH29 enzymes are still unknown. We describe here the identification of the catalyticnucleophile of the reaction in the α-l-fucosidase from S. solfataricus by reactivation with sodium azideof the mutant Asp242Gly that shows a 103-fold activity reduction on pNp−Fuc. The detailed stereochemicalanalysis 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 unambiguousassignment 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 applicabilityof this method.
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