Characterization of a new  -L-fucosidase isolated from the marine mollusk Pecten maximus that catalyzes the hydrolysis of  -L-fucose from algal fucoidan (Ascophyllum nodosum)

Abstract

Algal fucoidan is an alpha-L-fucose-based polysaccharide endowed with important biological properties for which the structure has not yet been fully elucidated. In an attempt to implement new enzymatic tools for structural study of this polysaccharide, we have found a fucosidase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on a fucoidan extracted from the brown algae Ascophyllum nodosum. We now report the purification and characterization of this alpha-L-fucosidase (EC 3.2.1.51). The enzyme was purified by three chromatographic steps, including an essential affinity chromatography based on the glycosidase inhibitor analog 6-amino-deoxymannojirimycin as the ligand. The purified alpha-L-fucosidase is a tetrameric glycoprotein of 200 kDa that hydrolyzes the synthetic substrate p-nitrophenyl alpha-L-fucopyranoside with a K(m) value of 650 microM. This enzyme has high catalytic activity (85 micromol x min(-1) x mg(-1)) compared with the other known fucosidases and also possesses an unusual thermal stability. The purified alpha-L-fucosidase is a retaining glycosidase. The activity of the purified fucosidase was determined on two structurally different fucoidans of the brown algae A. nodosum and Fucus vesiculosus to delineate glycosidic bond specificity. This report is to our knowledge the first demonstration of a fucosidase that can efficiently release alpha-L-fucose from fucoidan.

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

“…5b). Despite different oligomeric states being reported for many prokaryotic and eukaryotic GH29 enzymes (16,20,23,24, 27, 36), we did not find any evidence for oligomerization.…”

“…Depending on the specificity of ␣-L-fucosidases, they actively hydrolyze a variety of terminal fucosyl linkages typically in the form of ␣1,2 linkages to D-galactose (Gal), ␣1,3/4/6 linkages to N-acetylglucosamine (GlcNAc), ␣1,3 linkages to D-glucose (Glc) that are present in different natural substrates like milk oligosaccharides, plant cell wall polysaccharides, various glycoproteins, or glycolipids harboring various A, B, H, and Lewis blood group antigens or their analogs (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In addition, ␣-L-fucosidase isolated from the marine mollusk Pecten maximus has been reported to efficiently release fucose without extensive depolymerization of fucoidan from Ascophyllum nodosum (24), which contains alternating Fuc␣1-4Fuc and Fuc␣1-3Fuc backbone units (14). On the other hand, an exofucosidase from abalone (Haliotis gigantea) liver has been demonstrated to catalyze complete hydrolysis of fucoidan from brown alga Ecklonia cava (25).…”

Crystal Structure of Thermotoga maritima α-l-Fucosidase

“…1 ␣-L-Fucosidases have been shown to follow a double displacement mechanism with net retention of the anomeric configuration, as initially proposed for human liver ␣-L-fucosidase in 1987 (18) and recently confirmed by biochemical studies on ␣-L-fucosidases from Thermus sp. Y5 (19), the marine mollusc Pecten maximus (20), Sulfolobus solfataricus (21), and Thermotoga maritima (22).…”

Identification of the Catalytic Nucleophile of the Family 29 α-L-Fucosidase from Thermotoga maritima through Trapping of a Covalent Glycosyl-Enzyme Intermediate and Mutagenesis

“…Recent work has demonstrated that ␣-L-fucosidases are retaining enzymes; the hydrolyzed hemiacetal product bears the same stereochemical configuration as the original glycoside (11,12). In the vast majority of these cases this stereochemical outcome arises from a double displacement mechanism within the enzyme active site.…”