title: Analysis of the role of O-glycosylation in 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 with the aim of elucidating the role of glycosylation on the properties of the enzyme at the level of S160 residue. As a matter of fact, previous mass spectral analyses had led to the localization of a single O-glycosylation at this site. Recombinant expression and characterization of the rPoAbf mutant S160G was therefore performed. It was shown that the catalytic properties are slightly changed by the mutation, with a more evident modification of the Kcat and KM toward the synthetic substrate pN-glucopyranoside. More importantly, the mutation negatively affected the stability of the enzyme at various pHs and temperatures. Circular dichroism (CD) analyses showed a minimum at 210 nm for wild-type (wt) rPoAbf, typical of the beta-sheets structure, whereas this minimum is shifted for rPoAbf S160G, suggesting the presence of an unfolded structure. A similar behavior was revealed when wt rPoAbf was enzymatically deglycosylated. CD structural analyses of both the site-directed mutant and the enzymatically deglycosylated wild-type enzyme indicate a role of the glycosylation at the S160 residue in rPoAbf secondary structure stability.
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II," Complesso Universitario Monte S. Angelo, via Cinthia, Naples, Italy.
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, Naples, Italy.
authors.forename: Annabel
authors.initials: A
authors.lastname: Serpico
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II," Complesso Universitario Monte S. Angelo, via Cinthia, Naples, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II," Complesso Universitario Monte S. Angelo, via Cinthia, Naples, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Vinciguerra
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II," Complesso Universitario Monte S. Angelo, via Cinthia, Naples, Italy.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Faraco
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics
chemical_list: D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: arabinofuranosidase; fungus; glycosylation; lignocelluloses; site-directed mutagenesis