Analysis of the role of O‐glycosylation in GH51 α‐l‐arabinofuranosidase from Pleurotus ostreatus

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 K (cat) and K (M) 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.