title: Identification of the nucleophile catalytic residue of GH51 α-L-arabinofuranosidase from Pleurotus ostreatus.
abstract: In this study, the recombinant α-L-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis in order to identify the catalytic nucleophile residue. Based on bioinformatics and homology modelling analyses, E449 was revealed to be the potential nucleophilic residue. Thus, the mutant E449G of PoAbf was recombinantly expressed in Pichia pastoris and its recombinant expression level and reactivity were investigated in comparison to the wild-type. The design of a suitable set of hydrolysis experiments in the presence or absence of alcoholic arabinosyl acceptors and/or formate salts allowed to unambiguously identify the residue E449 as the nucleophile residue involved in the retaining mechanism of this GH51 arabinofuranosidase. (1)H NMR analysis was applied for the identification of the products and the assignement of their anomeric configuration.
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cinthia, 4, 80126, Naples, Italy. a.amore@unina.it.|National Renewable Energy Laboratory, Biosciences Center, 15013 Denver West Parkway, Golden, CO, 80401, USA. a.amore@unina.it.
authors.forename: Antonella
authors.initials: A
authors.lastname: Amore
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cinthia, 4, 80126, Naples, Italy. iadonisi@unina.it.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Iadonisi
authors.affiliation: CNRS, UMR7257, Aix-Marseille Universite, 163 Avenue de Luminy, Case 932, 13288, Marseille cedex 09, France. Florence.Vincent@afmb.univ-mrs.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Vincent
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cinthia, 4, 80126, Naples, Italy. vfaraco@unina.it.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Faraco
keywords: Arabinofuranosidase; Lignocellulose; Site-directed mutagenesis