Identification of an archaeal a-L-fucosidase encoded by an interrupted gene: production of a functional enzyme by mutations mimicking programmed -1 frameshifting

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a 1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-L-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the 1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed 1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-L-fucosidase (EC 3.2.1.51) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed 1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.