Identification of an archaeal alpha-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 ) 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.
Cobucci-Ponzano
Beatrice
B
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
Trincone
Antonio
A
Giordano
Assunta
A
Rossi
Mosè
M
Moracci
Marco
M
D001105:Archaea / Q000201:enzymology*; D001483:Base Sequence; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D016368:Frameshift Mutation; D016680:Genome, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D016415:Sequence Alignment; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology; D014158:Transcription, Genetic; D005644:alpha-L-Fucosidase / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D005644:alpha-L-Fucosidase