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

Abstract

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 approximately 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 1H-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.

Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases

“…More generally, we now have six families of GHs encompassing enzymes degrading sulfated polysaccharides: families GH16 (κ-carrageenases, keratanases), GH79 (heparanases), GH82 (ι-carrageenases), a new GH family specific for the degradation of sulfated fucans [46] and two unclassified enzymes, Bacillus circulans keratanase II [47] and P. carrageenovora λ-carrageenase (the present study). With the exception of the GH16 family, all these families encompass enzymes that interact exclusively with anionic polysaccharides.…”

Marine Polysaccharide Sulfatases

“…Fucanolytic enzymes include glycoside hydrolases and sulfatases. Gene sequences of fucanases have been validated by overexpression of active enzymes (Colin et al, 2006). However, although fucoidan sulfatase activity has been demonstrated in several bacterial strains, its sequence is still unknown and as for agarases and carrageenases in red algae, no sulfatase genes are predicted in brown algal genome (Cock et al, 2010).…”

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium, Formosa algae

“…Information regarding the specificity of fucoidanases, such as the type of glycosidic bond cleaved and the influence of the degree of sulfation of substrate on the catalytic activity, is scarce. The nucleotide sequences of the genes encoding fucoidanases and their deduced amino acid sequences have been published for the Mariniflexile fucanivorans SW5 [34] and Fucanobacter lyticus SN-1009 fucoidanases only [35], and technologically valuable sources of these enzymes have not yet been found.…”