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J. Biol. Chem., Vol. 278, Issue 17, 14622-14631, April 25, 2003

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Identification of an Archaeal -L-Fucosidase Encoded by an Interrupted Gene
PRODUCTION OF A FUNCTIONAL ENZYME BY MUTATIONS MIMICKING PROGRAMMED 1 FRAMESHIFTING^*,

Beatrice Cobucci-Ponzano, Antonio Trincone^§, Assunta Giordano^§, Mosè Rossi^¶, and Marco Moracci

From the  Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy, the ^§ Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy, and the ^¶ Dipartimento di Chimica Biologica Università di Napoli "Federico II," Via Mezzocannone 16, 80134 Naples, Italy

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 -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 -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 -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 -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.

This paper is dedicated to the memory of Eraldo Antonini, eminent biochemist, prematurely deceased 20 years ago, on March 19th, 1983.

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