Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene. Production of a functional enzyme by mutations mimicking programmed -1 frameshifting

J Biol Chem. 2003 Apr 25;278(17):14622-31. doi: 10.1074/jbc.M211834200. Epub 2003 Feb 4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaea / enzymology*
  • Base Sequence
  • Consensus Sequence
  • DNA, Complementary / genetics
  • Enzyme Stability
  • Frameshift Mutation*
  • Genome, Bacterial
  • Kinetics
  • Molecular Sequence Data
  • Mutation
  • Open Reading Frames
  • Sequence Alignment
  • Substrate Specificity
  • Sulfolobus / enzymology
  • Transcription, Genetic
  • alpha-L-Fucosidase / genetics*
  • alpha-L-Fucosidase / metabolism

Substances

  • DNA, Complementary
  • alpha-L-Fucosidase