Characterization of a novel endo-1,3-fucanase from Wenyingzhuangia fucanilytica within glycoside hydrolase family 168.

Sulfated fucan has attracted considerable research interest in recent years due to its diverse physiological activities. Fucanase is a critical tool for investigating sulfated fucans. In the present research, a novel endo-1,3-fucanase in the GH168 family, Fun168E, was identified within a sulfated fucan utilization loci from the genome of bacterium Wenyingzhuangia fucanilytica. Fun168E was a processive degrading enzyme and demonstrated a favorable thermostability. Ultra-performance liquid chromatography-mass spectrometry and NMR experiments demonstrated that Fun168E specifically hydrolyzed the α(1 → 3) linkages between Fucp2S and Fucp2S in sulfated fucan from Isostichopus badionotus, and α(1 → 3) linkages between Fucp2S and Fucp2,4S in sulfated fucan from Holothuria tubulosa. Fun168E could accommodate Fucp2S at subsite -1, and accept Fucp2,4S and Fucp2S at subsite +1. The discovery of this novel endo-1,3-fucanase would promote the utilization of sulfated fucans and their oligosaccharides in future applications.

The structure investigation of GH174 endo-1,3-fucanase revealed an unusual glycoside hydrolase fold.

Sulfated fucan has attracted increasing research interest due to its various biological activities. Endo-1,3-fucanases are favorable tools for structure investigation and structure-activity relationships establishment of sulfated fucan. However, the three-dimensional structure of enzymes from the GH174 family has not been disclosed, which hinders the understanding of the action mechanism. This study reports the first crystal structure of endo-1,3-fucanase from GH174 family (Fun174A) at a resolution of 1.60 Å. Notably, Fun174A exhibited an unusual distorted ß-sandwich fold, which is distinct from other known glycoside hydrolase folds. The conserved amino acid residues D119 and H154 were proposed as the catalytic residues in the family. Molecular docking suggested that Fun174A primarily recognized sulfated fucan through a series of polar amino acid residues around the substrate binding pocket. Furthermore, structural bioinformatics analysis suggested that the structural analogs of Fun174A may be extensively implicated in the bacterial metabolism of polysaccharides, which provided opportunities for the discovery of novel glycoside hydrolases. This study offers new insights into the structural diversity of glycoside hydrolases and will contribute to the establishment of a novel clan of glycoside hydrolases.

Structural investigation of Fun168A unraveling the recognition mechanism of endo-1,3-fucanase towards sulfated fucan.

BACKGROUND: Sulfated fucan has gained interest due to its various physiological activities. Endo-1,3-fucanases are valuable tools for investigating the structure and establishing structure-activity relationships of sulfated fucan. However, the substrate recognition mechanism of endo-1,3-fucanases towards sulfated fucan remains unclear, limiting the application of endo-1,3-fucanases in sulfated fucan research. SCOPE AND APPROACH: This study presented the first crystal structure of endo-1,3-fucanase (Fun168A) and its complex with the tetrasaccharide product, utilizing X-ray diffraction techniques. The novel subsite specificity of Fun168A was identified through glycomics and nuclear magnetic resonance (NMR). KEY FINDINGS AND CONCLUSIONS: The structure of Fun168A was determined at 1.92 Å. Residues D206 and E264 acted as the nucleophile and general acid/base, respectively. Notably, Fun168A strategically positioned a series of polar residues at the subsites ranging from -2 to +3, enabling interactions with the sulfate groups of sulfated fucan through salt bridges or hydrogen bonds. Based on the structure of Fun168A and its substrate recognition mechanisms, the novel subsite specificities at the -2 and +2 subsites of Fun168A were identified. Overall, this study provided insight into the structure and substrate recognition mechanism of endo-1,3-fucanase for the first time and offered a valuable tool for further research and development of sulfated fucan.

Biochemical characterization and cleavage specificities analyses of three endo-1,3-fucanases within glycoside hydrolase family 174.

Sulfated fucans have garnered extensive research interest in recent decades due to their varied bioactivity. Fucanases are important tools for investigating sulfated fucans. This study reported the bioinformatic analysis and biochemical properties of three GH174 family endo-1,3-fucanases. Wherein, Fun174Rm and Fun174Sb showed the highest optimal reaction temperature among the reported fucanases, and Fun174Sb possessed favorable thermostability and catalysis efficiency. Fun174Rm displayed a random endo-acting manner, while Fun174Ri and Fun174Sb hydrolyzed sulfated fucan in processive manners. UPLC-MS and NMR analyses confirmed that the three enzymes catalyze cleavage of the α(1 â†’ 3)-bonds between Fucp2S and Fucp2S in the sulfated fucan from Isostichopus badionotus. These enzymes demonstrated novel cleavage specificities, which could accept α-Fucp2S residues at subsites -1 and + 1. The acquiring of these biotechnological tools would be beneficial to the in-depth research of sulfated fucans.

Characterization of a novel endo-1,3-fucanase from marine bacterium Wenyingzhuangia fucanilytica reveals the presence of diversity within glycoside hydrolase family 168.

Sulfated fucans attract increasing research interests in recent decades for their various physiological activities. Fucanases are indispensable tools for the investigation of sulfated fucans. Herein, a novel GH168 family endo-1,3-fucanase was cloned from the genome of marine bacterium Wenyingzhuangia fucanilytica. The expressed protein Fun168D was a processive endo-acting enzyme. Ultra performance liquid chromatography-high resolution mass spectrum and NMR analyses revealed that the enzyme cleaved the α-1 â†’ 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2OSO3-) in sulfated fucan from Isostichopus badionotus, and α-1 â†’ 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2,4OSO3-) in sulfated fucan from Holothuria tubulosa. Fun168D would prefer to accept α-l-Fucp(2,4OSO3-) than α-l-Fucp(2OSO3-) at subsite +1, and could tolerate the absence of fucose residue at subsite +2. The novel cleavage specificity and hydrolysis pattern revealed the presence of diversity within the GH168 family, which would facilitate the development of diverse biotechnological tools for the molecule tailoring of sulfated fucan.

Sulfated fucan could serve as a species marker of sea cucumber with endo-1,3-fucanase as the essential tool.

In the past few decades, sulfated fucan from sea cucumber had attracted considerable interest owing to its abundant physiological activities. Nevertheless, its potential for species discrimination had not been investigated. Herein, particular attention was given to sea cucumber Apostichopus japonicus, Acaudina molpadioides, Holothuria hilla, Holothuria tubulosa, Isostichopus badionotus and Thelenota ananas to examine the feasibility of sulfated fucan as a species marker of sea cucumber. The enzymatic fingerprint suggested that sulfated fucan exhibited significant interspecific discrepancy and intraspecific stability, which revealed that sulfated fucan could serve as the species marker of sea cucumber, by utilizing the overexpressed endo-1,3-fucanase Fun168A and the ultra-performance liquid chromatography-high resolution mass spectrum. Moreover, oligosaccharide profile of sulfated fucan was determined. The oligosaccharide profile combined with hierarchical clustering analysis and principal components analysis further confirmed that sulfated fucan could serve as a marker with a satisfying performance. Besides, load factor analysis showed that the minor structure of sulfated fucan also contributed to the sea cucumber discrimination, besides the major structure. The overexpressed fucanase played an indispensable role in the discrimination, due to its specificity and high activity. The study would lead to a new strategy for species discrimination of sea cucumber based on sulfated fucan.

Characterization of an endo-1,3-fucanase from marine bacterium Wenyingzhuangia aestuarii: The first member of a novel glycoside hydrolase family GH174.

Sulfated fucans are important marine polysaccharides with various biological and biomedical activities. Fucanases are favorable tools to establish the structure-activity relationships of sulfated fucans. Herein, gene fun174A was discovered from the genome of marine bacterium Wenyingzhuangia aestuarii OF219, and none of the pre-defined glycosidic hydrolase domains were predicted in the protein sequence of Fun174A. Recombinant Fun174A demonstrated a low optimal reaction pH at 5.5. It might degrade sulfated fucans in an endo-processive manner. Glycomics and NMR analyses proved that it specifically hydrolyzed α-1,3-l-fucoside bonds between 2-O-sulfated and non-sulfated fucose residues in the sulfated fucan from sea cucumber Isostichopus badionotus. D119, E120 and E218 were critical for the activity of Fun174A, as identified by site-directed mutagenesis. Three homologs of Fun174A were confirmed to exhibit endo-1,3-fucanase activities. The novelty on sequences of Fun174A and its homologs reveals a new glycoside hydrolase family, GH174.

The Discovery of the Fucoidan-Active Endo-1→4-α-L-Fucanase of the GH168 Family, Which Produces Fucoidan Derivatives with Regular Sulfation and Anticoagulant Activity.

Sulfated polysaccharides of brown algae, fucoidans, are known for their anticoagulant properties, similar to animal heparin. Their complex and irregular structure is the main bottleneck in standardization and in defining the relationship between their structure and bioactivity. Fucoidan-active enzymes can be effective tools to overcome these problems. In the present work, we identified the gene fwf5 encoding the fucoidan-active endo-fucanase of the GH168 family in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The biochemical characteristics of the recombinant fucanase FWf5 were investigated. Fucanase FWf5 was shown to catalyze the endo-type cleavage of the 1→4-O-glycosidic linkages between 2-O-sulfated α-L-fucose residues in fucoidans composed of the alternating 1→3- and 1→4-linked residues of sulfated α-L-fucose. This is the first report on the endo-1→4-α-L-fucanases (EC 3.2.1.212) of the GH168 family. The endo-fucanase FWf5 was used to selectively produce high- and low-molecular-weight fucoidan derivatives containing either regular alternating 2-O- and 2,4-di-O-sulfation or regular 2-O-sulfation. The polymeric 2,4-di-O-sulfated fucoidan derivative was shown to have significantly greater in vitro anticoagulant properties than 2-O-sulfated derivatives. The results have demonstrated a new type specificity among fucanases of the GH168 family and the prospects of using such enzymes to obtain standard fucoidan preparations with regular sulfation and high anticoagulant properties.

Systematic comparison of eight methods for preparation of high purity sulfated fucans extracted from the brown alga Pelvetia canaliculata.

Sulfated fucans from brown algae are a heterogeneous group of biologically active molecules. To learn more on their structure and to analyze and exploit their biological activities, there is a growing need to develop reliable and cost effective protocols for their preparation. In the present study, a brown alga Pelvetia canaliculata (Linnaeus) was used as a rich source of sulfated fucans. Sulfated fucan preparation methods included neutral and acidic extractions followed by purification with activated charcoal (AC), polyvinylpolypyrrolidone (PVPP), or cetylpyridinium chloride (CPC). Final products were compared in terms of yield, purity, monosaccharide composition and molecular weight. Acidic extractions provided higher yields compared to neutral ones, whereas the AC purification provided sulfated fucan products with the highest purity. Mass spectrometry analyses were done on oligosaccharides produced by the fucanase MfFcnA from the marine bacterium Mariniflexille fucanivorans. This has provided unique insight into enzyme specificity and the structural characteristics of sulfated fucans.

In Vitro Anti-Orthohantavirus Activity of the High-and Low-Molecular-Weight Fractions of Fucoidan from the Brown Alga Fucus evanescens.

The Hantaan orthohantavirus (genovariant Amur-AMRV) is a rodent-borne zoonotic virus; it is the causative agent of haemorrhagic fever with renal syndrome in humans. The currently limited therapeutic options require the development of effective anti-orthohantavirus drugs. The ability of native fucoidan from Fucus evanescens (FeF) and its enzymatically prepared high-molecular-weight (FeHMP) and low-molecular-weight (FeLMP) fractions to inhibit different stages of AMRV infection in Vero cells was studied. The structures of derivatives obtained were determined using nuclear magnetic resonance (NMR) spectroscopy. We found that fucoidan and its derivatives exhibited significant antiviral activity by affecting the early stages of the AMRV lifecycle, notably virus attachment and penetration. The FeHMP and FeLMP fractions showed the highest anti-adsorption activity by inhibiting AMRV focus formation, with a selective index (SI) > 110; FeF had an SI of ~70. The FeLMP fraction showed a greater virucidal effect compared with FeF and the FeHMP fraction. It was shown by molecular docking that 2O-sulphated fucotetrasaccharide, a main component of the FeLMP fraction, is able to bind with the AMRV envelope glycoproteins Gn/Gc and with integrin ß3 to prevent virus-cell interactions. The relatively small size of these sites of interactions explains the higher anti-AMRV activity of the FeLMP fraction.

Utilizing heterologously overexpressed endo-1,3-fucanase to investigate the structure of sulfated fucan from sea cucumber (Holothuria hilla).

Sea cucumber sulfated fucan (SC-FUC) attracted increasing interests in the recent decades. Endo-1,3-fucanase has been employed in the structural clarification and structure-function relationship investigations of SC-FUC. Nevertheless, the preparation of wild-type endo-1,3-fucanase is costly and time-consuming, which hinders its further utilization. In this study, a heterologously overexpressed endo-1,3-fucanase (FunA) was introduced into structural identification of SC-FUC. FunA was efficiently prepared within one day and utilized in the investigation of sulfated fucan from sea cucumber Holothuria hilla (Hh-FUC). By using enzymatic degradation, glycomics and NMR analysis, the major structure of Hh-FUC was identified to be composed of a tetrasaccharide repeating unit →3-α-l-Fucp-1 â†’ 3-α-l-Fucp2,4(OSO3-)-1 â†’ 3-α-l-Fucp2(OSO3-)-1 â†’ 3-α-l-Fucp2(OSO3-)-1→. Due to the efficient acquisition of enzyme and the superior oligosaccharide recovery, 0.6 mL of E. coli broth and 10 mg of Hh-FUC were sufficient for the structural identification. The results demonstrated the superiority of heterologously overexpressed fucanase over its wild-type enzyme in structural investigation of sulfated fucan.

Discovery and Characterization of an Endo-1,3-Fucanase From Marine Bacterium Wenyingzhuangia fucanilytica: A Novel Glycoside Hydrolase Family.

Sulfated fucans are important marine polysaccharides widely distributed in brown algae and echinoderms, which gained increasing research interest for their various biological and biomedical activities. Fucanases could serve as tools in the bioconversion and structural investigation of sulfated fucans. A few gene-defined endo-1,4-fucanases have been characterized, while the sequence of endo-1,3-fucanase remain unstudied. Here, an endo-1,3-fucanase gene funA was screened from the genome of marine bacterium Wenyingzhuangia fucanilytica CZ1127T using transcriptomics. None of the previously reported glycoside hydrolase domains were predicted in the enzyme FunA, which hydrolyzed sulfated fucans in a random endo-acting manner. Ultrahigh performance size exclusion chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that FunA specifically cleaves α-1,3 glycosidic linkage between 2-O-sulfated and non-sulfated fucose residues. FunA exhibited transglycosylating activity with glycerin, methanol, and L-fucose as acceptors. D206 and E264 were critical for the functioning of FunA as identified by the site-directed mutagenesis. Another five homologs of FunA were confirmed to possess endo-1,3-fucanase activities. This is the first report on the sequence of endo-1,3-fucanase. The novelty of FunA and its homologs in sequences and activity shed light on a novel glycoside hydrolase family, GH168.

Discovery and screening of novel metagenome-derived GH107 enzymes targeting sulfated fucans from brown algae.

Sulfated fucans, often denoted as fucoidans, are highly variable cell wall polysaccharides of brown algae, which possess a wide range of bioactive properties with potential pharmaceutical applications. Due to their complex architecture, the structures of algal fucans have until now only been partly determined. Enzymes capable of hydrolyzing sulfated fucans may allow specific release of defined bioactive oligosaccharides and may serve as a tool for structural elucidation of algal walls. Currently, such enzymes include only a few hydrolases belonging to the glycoside hydrolase family 107 (GH107), and little is known about their mechanistics and the substrates they degrade. In this study, we report the identification and recombinant production of three novel GH107 family proteins derived from a marine metagenome. Activity screening against a large substrate collection showed that all three enzymes degraded sulfated fucans from brown algae in the order Fucales. This is in accordance with a hydrolytic activity against α-1,4-fucosidic linkages in sulfated fucans as reported for previous GH107 members. Also, the activity screening gave new indications about the structural differences in brown algal cell walls. Finally, sequence analyses allowed identification of the proposed catalytic residues of the GH107 family. The findings presented here form a new basis for understanding the GH107 family of enzymes and investigating the complex sulfated fucans from brown algae. DATABASE: The assembled metagenome and raw sequence data is available at EMBL-EBI (Study number: PRJEB28480). Sequences of the GH107 fucanases (Fp273, Fp277, and Fp279) have been deposited in GenBank under accessions MH755451-MH755453.

Fucoidanases.

In recent years, the research of fucoidans has steadily increased. The interest in these substances is due to their various biological activities. Despite a wide range of biological activity and the lack of oral toxicity, fucoidans remain relatively unexploited as a source of medicines because of their heterogeneity. Enzymes that degrade polyanionic polysaccharides are widely used for establishing their structures and structure-activity relationships. Sometimes, to obtain preparations of polysaccharides with standard characteristics, for example, medicines and food supplements, enzymatic treatment can be also applied. Only a few sources of enzymes with fucoidanase activity have been described, and only a few studies regarding the isolation and characterization of fucoidanases have been performed. The data on the specificity of fucoidanases: the type of cleaved glycoside bond, the relation between catalytic activity and the degree of substrate sulfation are scarce. The review summarizes achievements in the research of fucoidanases, mechanisms of enzymatic degradation of fucoidans, as well as of structures of sulfated fucooligosaccharides obtained under the action of fucoidanases.