Cloning and biochemical characterization of the fucanase FcnA: Definition of a novel glycoside hydrolase family specific for sulfated fucans

Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an alpha-l-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared similar to 25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and H-1-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the alpha-(1 -> 4) glycosidic linkages within the blocks of repeating motifs [-> 4)-alpha-l-fucopyranosyl-2,3-disulfate-(1 -> 3)-alpha-l-fucopyranosyl-2-sulfate-(1 ->]n.