The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides

Abstract

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

Keywords: FTIR; Fucus evanescens; Sargassum mcclurei; T9SS; calcium dependency; sulfation.

Figure 1

Predicted protein domain structures of Fhf2 and Fhf2Δ484. Numbering corresponds to amino acids in the full-length Fhf2 sequence. Predicted domains are indicated with the amino acid numbers and in colors. Grey: secretion signal peptide, green: D1 domain; cyan: cadherin-like superfamily domain (IPR015919); yellow: a secretion system C-terminal sorting domain (IPR026444). Fhf2* is the full-length gene for heterologous expression in Escherichia coli devoid of the N-terminal signal peptide as well as the C-terminal T9SS domain. The C-terminal deletion mutant Fhf2Δ484 only contains the predicted catalytic D1 domain.

Figure 2

3D homology model of Fhf2. The putative N-terminal catalytic domain D1 is shown in green with the catalytic residues Asp227 and His297 as well as the conserved Tyr139 and Trp350 highlighted in pink; the C-terminal domains C1 and C2 are presented in blue and orange, respectively. The four predicted Ca²⁺ ions are indicated by cyan spheres and the predicted Na⁺ ion by a purple sphere. (A) The 3D model presented as cartoon, showing the beta-barrel fold of the predicted C1 and 2 domains as well as the beta-barrel fold surrounding the active site. (B) A zoom of the active site, nicely illustrating the active site amino acids. (C) A sphere model, showing the grove extending from the active site. (D) Electrostatic surface of the Fhf2 homology model: blue color indicates positively charged surface areas, while red color indicates negatively charged surface areas.

Figure 3

Purification and enzyme activity of Fhf2 and Fhf2Δ484. SDS-PAGE and Western blot of Fhf2 (A,B respectively) and Fhf2Δ484 (C,D respectively). (1) Fhf2 protein with the expected molecular weight of 98 kDa, including other protein bands with lower molecular weight, visible in the SDS-PAGE in (A), but not in the western blot in (B). (2) Purified Fhf2Δ484 protein with the expected molecular weight of 46 kDa and no other proteins visible. M) Protein marker. (E) Fucoidanase activity by C-PAGE of Fhf2 and Fhf2Δ484 on fucoidan fraction 2 extracted from Fucus evanescens (FeF2). (St) oligosaccharide products of the enzymatic reaction of FFA2 on fucoidan from F. evanescens. Reaction conditions were 0.9% substrate, 0.3 mg/ml enzyme, 10 mM Tris-HCl pH 7.4, 10 mM Ca²⁺, 125 mM NaCl, 35°C for 24 h.

Figure 4

C-PAGE of Fhf2∆484 activity on fucoidans from different brown seaweed species. (−) indicates control substrate and (+) Fhf2∆484 enzymatic reactions on fucoidans from different seaweed species F. evanescens (Fe), F. vesiculosus (Fv), S. mcclurei (Sm), S. polycystum (Sp) and T. ornata (To). (St) positive control reaction of FFA2 on F. evanescens fucoidan. Reaction conditions were 0.9% substrate, 0.3 mg/ml enzyme, 10 mM Tris-HCl pH 7.4 and 125 mM NaCl at 35°C for 24 h.

Figure 5

Time course of Fhf2∆484 fucoidanase on fucoidan from F. evanescens. The Fhf2∆484 activity was measured at different time points during 0–48 h of reaction and visualized by (A) C-PAGE or (B) HPSEC. (FeF2) fucoidan substrate from F. evanescens, (St) positive control, reaction of FFA2 on F. evanescens fucoidan. Tentatively suggested oligosaccharide sizes are indicated (DP4-10). Reaction conditions were 0.9% substrate, 0.3 mg/ml enzyme, 10 mM Tris-HCl pH 7.4, 125 mM NaCl at 35°C for varying reaction times. HPSEC data are shown in logarithmic scale and the molecular weight is in Da.

Figure 6

Fhf2∆484 activity under different reaction conditions shown by C-PAGE. Activity of Fhf2∆484 on fucoidan from F. evanescens (FeF2) under the influence of different (A) pH (at 35°C), (B) temperature (at pH 8), (C) divalent cations at 10 mM (pH 8 and 37°C), (*) indicates the activity after addition of EDTA, e.g., without presence of any divalent cations, and (D) NaCl concentration dependency (at pH 8 and 37°C). (St) positive control reaction of FFA2 on F. evanescens fucoidan. General reaction conditions were 0.9% substrate, 0.3 mg/ml enzyme, 10 mM Tris-HCl and 4 h reaction time.

Figure 7

Thermostability of Fhf2∆484. Fhf2∆484 was incubated without substrate for the indicated time periods, before the activity was determined on fucoidan from F. evanescens. (A) 37°C, (B) 40°C, and (C) 45°C. (St) positive control reaction of FFA2 on F. evanescens fucoidan. Reaction conditions: 0.9% substrate, 0.3 mg/ml enzyme, 10 mM Tris-HCl pH 8, 100 mM NaCl, 37°C, 4 h reaction time.

Figure 8

Characterization of the Fhf2∆484 hydrolysis products. (A) Enzymatic reaction of Fhf2Δ484 on fucoidan from F. evanescens shown by C-PAGE. (FeF2) control fucoidan substrate from F. evanescens. (R) Enzymatic reaction of Fhf2Δ484 on fucoidan from F. evanescens, (HMWF) high molecular weight fucoidan and (LMWF) low molecular weight fucoidan, (HMWF*) second enzyme treatment of HMWF with Fhf2Δ484. (St) positive control reaction of FFA2 on F. evanescens fucoidan. (B) ¹H NMR spectra of F. evanescens fucoidan substrate (blue), high molecular weight fraction (black) and low molecular weight fraction (green) after hydrolysis by Fhf2Δ484.

Figure 9

Chemical fine-structures of the LMWF oligosaccharides released by Fhf2Δ484 from fucoidan from F. evanescens. (A) General molecular structure containing 2-sulfated residues along 2-sulfated/3-acetylated, 2-sulfated/4-acetylated and 2-,4-disulfated residues; (B) Molecular structure of a main product; (C) Molecular structure of the substrate indicating the endo-1,4 cleavage site that can tolerate acetylated fucosyl units in the −1 position and 2,4-disulfated fucosyl units nearby; (D) 2,4-disulfated fucose occurs to some degree at non-terminal sites and (E) 2-sulfated and 4 acetylated fucose occurs to some degree at the reducing end.

Figure 10

¹H-¹³C NMR spectrum and molecular structure of the high molecular weight product obtained upon F. evanescens fucoidan degradation with Fhf2Δ484.

Figure 11

FTIR spectral evolution profiles for the Fhf2Δ484 endo-fucoidanase. The spectral evolution changes (30 spectra) in response to the enzyme concentration. The background spectra of buffer and substrate were subtracted. 2% w/v of fucoidans from F. evanescens was used at increasing enzyme dosages: (A) 1.98 μM, (B) 3.3 μM, (C) 4.83 μM, (D) 7.69 μM, (E) 9.45 μM, and (F) 18.02 μM.

Figure 12

PARAFAC first component scores versus enzyme dosage plotted to build calibrations for Fhf2Δ484 on F. evanescens. The straight line, with the equation of Y = −0.0002 X + 0.0015, was fitted with the data. R² = 0.95. Due to sign ambiguity in component analysis, the sign of the slope of the fitted calibration is not linked to substrate or product character.