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Fucosylated oligosaccharides play several biologically relevant roles. They are naturally present in human milk which offers to infants short- and long-term health benefits. These compounds can be obtained also by enzymatic synthesis. In this work, the effects of pH and temperature on hydrolytic and transfucosylation activities of α-L-fucosidase from Thermotoga maritima were evaluated. The optimal pH for the enzyme-catalyzed hydrolysis was found in a range from 6 to 8 while the highest conversions for transfucosylation reactions were observed within the range of 7-10. The best temperature for both enzymatic activities was 95 °C. Fucosylated oligosaccharides were synthesized with the highest productivity of 3.54 mM/h at pH 8 and 95 °C. Overall, optimization of the conditions of transfucosylation reaction catalyzed by the α-L-fucosidase from Thermotoga maritima allowed for higher yields of fucosylated oligosaccharides as well as shorter reaction time and a lower concentration of the employed enzyme.

The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family’s large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/proteins generated by the action of endo-β- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs), we observed two distinct groups of B. pseudocatenulatum, isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO-binding specificities of the family 1 solute-binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain's distinct HMO consumption pattern. Taken together, these data indicate the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breastfed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies key gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

α- l -Fucosidase (AFu) is an important biomarker for early diagnosis of hepatocellular carcinoma and fucosidosis. In this study, a novel, sensitive and selective polymer dots (PDs) fluorescent sensing strategy based on inner filter effect was firstly developed for AFu activity determination. The PDs with a high quantum yield of 53.6% were synthesized by a mild one-pot method. The detection mechanism of this strategy was based on inner filter effect between PDs and p-nitrophenol (PNP) which was the hydrolyzate of 4-nitrophenyl-α- l -fucopyranoside (PNPF) catalyzed by AFu. The absorption of PNP overlapped the fluorescence excitation spectrum of the PDs, which resulted in a fluorescence quenching or weakening of PDs. The sensing system showed a good linear relationship within 0.01-0.9 U L−1 and provided a low AFu detection limit of 0.001 U L−1 (S/N = 3). This PDs sensor were successfully applied for the determination of AFu in human serum samples.

Techniques for synthesis of bespoke oligosaccharides currently lag behind those for other biopolymers such as polypeptides and polynucleotides, in part because of the paucity of satisfactory enzymatic tools to perform the synthetic reactions. One promising avenue of development for this problem is glycoside hydrolase enzymes with mutated nucleophile residues (called glycosynthases), which retain some elements of their native specificity and work with cheaply available substrates. However, the mechanistic underpinnings of this class of enzymes are not yet well-understood, and what few atomistic studies have been conducted have found different reaction pathways. In this paper, we describe the first unbiased computational study of the mechanism of a GH29 glycosynthase enzyme, Thermotoga maritima α-L-fucosidase (TmAfc) D224G. We find a single-step endothermic reaction step with an oxocarbenium-like transition state, demonstrating how stabilization of this transition state structure (which is common to many retaining glycoside hydrolases) can be repurposed in mutant enzymes to perform synthesis instead of hydrolysis. Our results are consistent with previous experimental observations and help both to clarify the mechanism of the existing single-mutant and to provide directions for further engineering of this and other glycosynthases.

2′-Fucosyllactose (2′-FL) is one of the nutrient ingredients in human milk, which has various beneficial health effects. α- l -fucosidase is a biotechnological tool for 2′-FL preparation. Here, a novel and efficient α- l -fucosidase OUC-Jdch16 from the fucoidan-digesting strain Flavobacterium algicola 12076 was heterologously expressed and applied to produce 2′-FL in vitro. OUC-Jdch16 belongs to glycoside hydrolases (GH) family 29 and exhibits the highest 4-nitrophenyl-α- l -fucopyranoside-hydrolyzing activity at 25 °C and pH 6.0. OUC-Jdch16 could catalyze the synthesis of 2′-FL via transferring the fucosyl residue from pNP-α-fucose to lactose. Under the optimal transfucosylation conditions, the yield of the transfucosylation product reached 84.82% and 92.15% (mol/mol) from pNP-α-fucose within 48 h and 120 h, respectively. Moreover, OUC-Jdch16 was capable of transferring the fucosyl residue to other glycosyl receptors with the generation of novel fucosylated compounds. This study demonstrated that OUC-Jdch16 could be a promising tool to prepare 2′-FL and other novel glycosides.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-l-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-l-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1–3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3′-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

Human milk is the optimal nutrition source for infants, and oligosaccharides represent the third most abundant component in milk after lactose and fat. Human milk oligosaccharides (HMO) are favorable macromolecules which are, interestingly, indigestible by the infant but serve as substrates for bacteria. Hypothesizing that the maternal diet itself might influence HMO composition, we sought to directly determine the effect maternal diet on HMO and the milk bacteria. Employing a human cross-over study design, we demonstrate that distinct maternal dietary carbohydrate and energy sources preferentially alter milk concentrations of HMO, including fucosylated species. We find significant associations between the concentration of HMO-bound fucose and the abundance of fucosidase (a bacterial gene that digests fucose moieties) harbored by milk bacteria. These studies reveal a successive mechanism by which the maternal diet during lactation alters milk HMO composition, which in turn shapes the functional milk microbiome prior to infant ingestion.

Fucoidan oligosaccharides possesses diverse physicochemical and biological activities. Specific glycoside hydrolases are valuable tools for degrading polysaccharides to produce oligosaccharides. In this study, BcFucA, a novel fucosidase belonging to GH95 family from Bacillus cereus 2–8, was cloned into pET21a vector, expressed in E. coli BL21 (DE3) and characterized. The protein consists of 1136 amino acid residues encoded by 3411 bases and has a molecular weight of 125.35 kDa. The optimal temperature and pH of this enzyme are 50 °C and pH 4.0. In addition, this study showed that the unknown function domain (encoding Lys261-Thr681) defined as a linker is quite important for its activity. The obtained novel enzyme BcFucA will contribute to the effective degradation of fucoidan and future industrial applications.

The pinewood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of the pine wilt disease (PWD), enters above-ground parts of the tree, migrates through the resin canals and feeds on plant cells causing extensive damage. In order to penetrate the cell wall and establish a parasitic relationship with host trees, the PWN needs to secretea mixture of active cell wall degrading enzymes. In maritime pine, Pinus pinaster, which is high susceptible to PWN, xyloglucan is the major hemicellulosic polysaccharide in primary cells. The xyloglucan backbone is susceptible to hydrolysis by numerous endoglucanases, some of them specific to xyloglucan. However, to completely degrade xyloglucan, all substitutions on the glucan backbones must be released, and l-fucose residues in xyloglucan branches are released by &alpha, l-fucosidases. In the present study, the molecular characterization of two &alpha, l-fucosidases found in PWN secretome was performed. Moreover, a novel &alpha, l-fucosidase was identified and its cDNA and gene sequence were determined. The three-dimensional structures of these &alpha, l-fucosidases were predicted and the transcript levels were analyzed, thus providing new insights into fundamental PWN biology and the possible role of these proteins as cell wall degrading enzymes.

Fucosyllactoses have gained much attention owing to their multiple functions, including prebiotic, immune, gut, and cognition benefits. In this study, human milk oligosaccharide (HMO) 2'-fucosyllactose (α-L-Fuc-(1,2)-D-Galβ-1,4-Glu, 2'FL) and its isomer 3'-fucosyllactose (α-L-Fuc-(1,3)-D-Galβ-1,4-Glu, 3'FL) with potential prebiotic effect were synthesized efficiently by a novel recombinant α-L-fucosidase. An α-L-fucosidase gene (PbFuc) from Pedobacter sp. CAU209 was successfully cloned and expressed in Escherichia coli (E. coli). The deduced amino acid sequence shared the highest identity of 36.8% with the amino sequences of other reported α-L-fucosidases. The purified α-L-fucosidase (PbFuc) had a molecular mass of 50 kDa. The enzyme exhibited specific activity (26.3 U/mg) towards 4-nitrophenyl-α-L-fucopyranoside (pNP-FUC), 3'FL (8.9 U/mg), and 2'FL (3.4 U/mg). It showed the highest activity at pH 5.0 and 35 °C, respectively. PbFuc catalyzed the synthesis of 3'FL and 2'FL through a transglycosylation reaction using pNP-FUC as donor and lactose as acceptor, and total conversion ratio was up to 85% at the optimized reaction conditions. The synthesized mixture of 2'FL and 3'FL promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus NRRL B-548, L. casei subsp. casei NRRL B-1922, L. casei subsp. casei AS 1.2435, and Bifidobacterium longum NRRL B-41409. However, the growths of E. coli ATCC 11775, S. enterica AS 1.1552, L. monocytogenes CICC 21635, and S. aureus AS 1.1861 were not stimulated by the mixture of 2'FL and 3'FL. Overall, our findings suggest that PbFuc possesses a great potential for the specific synthesis of fucosylated compounds.Key Points• A novel α-L-fucosidase (PbFuc) from Pedobacter sp. was cloned and expressed.• PbFuc showed the highest hydrolysis activity at pH 5.0 and 35 °C, respectively.• It was used for synthesis of 3'-fucosyllactose (3'FL) and 2'-fucosyllactose (2'FL).• The mixture of 3'FL and 2'FL promoted the growth of some Lactobacillus sp. and Bifidobacteria sp.

Fucosylated oligosaccharides, such as 2'-fucosyllactose in human milk, have important biological functions such as prebiotics and preventing infection. In this work, the effect of an acceptor substrate (lactose) and the donor substrate 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) on the synthesis of a fucosylated trisaccharide was studied in a transglycosylation reaction using α-L-fucosidase from Thermotoga maritima. Conducting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), it was demonstrated that synthesized oligosaccharide corresponded to a fucosylated trisaccharide, and high-performance liquid chromatography (HPLC) of the hydrolyzed compound confirmed it was fucosyllactose. As the concentration of the acceptor substrate increased, the concentration and synthesis rate of the fucosylated trisaccharide also increased, and the highest concentration obtained was 0.883 mM (25.2% yield) when using the higher initial lactose concentration (584 mM). Furthermore, the lower donor/acceptor ratio had the highest synthesis, so at the molar ratio of 0.001, a concentration of 0.286 mM was obtained (32.5% yield).

Core fucosylation is the attachment of an α-1,6-fucose moiety to the innermost N-acetyl glucosamine (GlcNAc) in N-glycans in mammalian systems. It plays a pivotal role in modulating the structural and biological functions of glycoproteins including therapeutic antibodies. Yet, few α-l-fucosidases appear to be capable of removing core fucose from intact glycoproteins. This paper describes a comparative study of the substrate specificity and relative activity of the human α-l-fucosidase (FucA1) and two bacterial α-l-fucosidases, the AlfC from Lactobacillus casei and the BfFuc from Bacteroides fragilis. This study was enabled by the synthesis of an array of structurally well-defined core-fucosylated substrates, including core-fucosylated N-glycopeptides and a few antibody glycoforms. It was found that AlfC and BfFuc could not remove core fucose from intact full-length N-glycopeptides or N-glycoproteins but could hydrolyze only the truncated Fucα1,6GlcNAc-peptide substrates. In contrast, the human α-l-fucosidase (FucA1) showed low activity on truncated Fucα1,6GlcNAc substrates but was able to remove core fucose from intact and full-length core-fucosylated N-glycopeptides and N-glycoproteins. In addition, it was found that FucA1 was the only α-l-fucosidase that showed low but apparent activity to remove core fucose from intact IgG antibodies. The ability of FucA1 to defucosylate intact monoclonal antibodies reveals an opportunity to evolve the human α-l-fucosidase for direct enzymatic defucosylation of therapeutic antibodies to improve their antibody-dependent cellular cytotoxicity.

The protective function of the intestinal mucosa largely depends on carbohydrate moieties that as a part of glycoproteins and glycolipids form the epithelial glycocalyx or are secreted as mucins. Modifications of their expression can be induced by an altered intestinal microenvironment and have been associated with inflammatory disorders and colorectal cancer. Given the influence of dietary factors on the gut ecosystem, here we have investigated whether a long term feeding on a starch-rich diet can modulate the glucidic profile in the colonic mucosa of rats. Animals were divided into two groups and maintained for 9 months at different diets: one group was fed a standard diet, the second was fed a starch-enriched diet. Samples of colonic mucosa, divided in proximal and distal portions, were processed for microscopic analysis. Conventional stainings and lectin histochemistry were applied to identify acidic glycoconjugates and specific sugar residues in oligosaccharide chains, respectively. Some lectins were applied on adjacent sections after sialidase/fucosidase digestion, deacetylation, and oxidation to characterize either terminal dimers or sialic acid acetylation. An increase in sulfomucins was found to be associated with the starch-enriched diet that affected also the expression of several sugar residues as well as fucosylated and sialylated sequences in both proximal and distal colon. Although the mechanisms leading to such a modulation are at present unknown, either an altered intestinal microbiota or a dysregulation of glycosylation patterns might be responsible for the types and distribution of changes in the glucidic profile here observed.

Glycosylation is a post-translational modification that is an essential element in cell signaling and neurodevelopmental pathway regulation. Glycan attachment can influence the tertiary structure and molecular interactions of glycosylated substrates, adding an additional layer of regulatory complexity to functional mechanisms underlying central cell biological processes. One type of enzyme-mediated glycan attachment, fucosylation, can mediate glycoprotein and glycolipid cell surface expression, trafficking, secretion, and quality control to modulate a variety of inter- and intracellular signaling cascades. Building on prior reports of glycosylation abnormalities and evidence of dysregulated glycosylation enzyme expression in schizophrenia, we examined the protein expression of 5 key fucose-modifying enzymes: GDP-fucose:protein O -fucosyltransferase 1 (POFUT1), GDP-fucose:protein O -fucosyltransferase 2 (POFUT2), fucosyltransferase 8 (FUT8), fucosyltransferase 11 (FUT11), and plasma α- l -fucosidase (FUCA2) in postmortem superior temporal gyrus of schizophrenia (N = 16) and comparison (N = 14) subjects. We also used the fucose binding protein, Aleuria aurantia lectin (AAL), to assess α-1,6-fucosylated N -glycoprotein abundance in the same subjects. In schizophrenia, we found increased expression of POFUT2, a fucosyltransferase uniquely responsible for O -fucosylation of thrombospondin-like repeat domains that is involved in a non-canonical endoplasmic reticulum quality control pathway. We also found decreased expression of FUT8 in schizophrenia. Given that FUT8 is the only α-1,6-fucosyltransferase expressed in mammals, the concurrent decrease in AAL binding in schizophrenia, particularly evident for N -glycoproteins in the ~ 52–58 kDa and ~ 60–70 kDa molecular mass ranges, likely reflects a consequence of abnormal FUT8 expression in the disorder. Dysregulated FUT8 and POFUT2 expression could potentially explain a variety of molecular abnormalities in schizophrenia.

Although core xylose on glycoproteins has been implicated in allergy, infection and other biological processes, research on core xylose modification is rare. The lack of a β-d-xylosidase that can catalytically remove the core xylose directly from glycoproteins is a reason for this. Through functional genomic analysis, we identified a glycoprotein core xylosidase and named it gpcXase I. gpcXase I is located immediately downstream of glycoprotein core fucosidase cFase I in Elizabethkingia meningoseptica. These two genes form a functional operon for glycoprotein core modifications. Three acidic residues (Asp-200, Asp-304 and Glu-649) were identified as key catalytic sites for gpcXase I activity, suggesting a unique triacdic mechanize for its activity. Asp-200 was identified a novel and essential base catalysts in the catalytic process, Asp-304 and Glu-649 was function as catalytic nucleophiles and acid catalysts, respectively. In addition, IgE-specific reactions were detected in 55% of serum samples collected from 40 allergic patients, and the reactions were significantly attenuated by removal of the core xylose of the allergen by treatment with gpcXase I. gpcXase I is a novel tool for basic and clinical glycomics.

Fucosyl-oligosaccharides are natural prebiotics that promote the growth of probiotics in human gut and stimulate the innate immune system. In this work, the release of &alpha, lfucosidase by Lactobacillus rhamnosus GG, and the use of this enzyme for the synthesis of fucosyl-oligosaccharides were investigated. Since &alpha, lfucosidase is a membrane-bound enzyme, its release from the cells was induced by addition of 4-nitrophenyl-&alpha, l-fucopyranoside (pNP-Fuc). Enzyme activity associated with the cell was recovered at 78% of its total activity. Fucosyl-oligosaccharides where synthesized using &alpha, l-fucosidase extract and pNP-Fuc as donor substrate, and D-lactose or D-lactulose as acceptor substrates, reaching a yield up to 25%. Fucosyllactose was obtained as a reaction product with D-lactose, and its composition was confirmed by mass spectrometry (MALDI-TOF MS). It is possible that the fucosyl-oligosaccharide synthesized in this study has biological functions similar to human milk oligosaccharides.

2′-Fucosyllactose (2′-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2′-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2′-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l -fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli . The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni 2+ affinity chromatography. The purified α1,2-fucosidase and l -fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2′-FL in a large number of samples simultaneously. This method can be used to quantify 2′-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2′-FL.

The characterization of a recombinant glycosidase can be done with commercially available substrates, which enable testing of enzyme functionality and determination of linkage specificity. Colorimetric assays with p-nitrophenyl substrates provide a relatively simple and fast way of screening conditions which could affect enzyme activity (buffer, pH, ion dependence, temperature). These substrates are useful for the determination of activity optima and the characterization of basic activity parameters. However, testing for linkage specificity should be performed on more complex sugars presenting a range of different glycosidic bonds and might need more sophisticated methods of analysis. This protocol provides comprehensive instructions on how to perform an initial characterization of your glycosidase using a recombinant α-L-fucosidase as an example.

An α- l -fucosidase (Pap-Alf) was purified from the pancreas of a starfish Patiria pectinifera by ammonium sulfate precipitation followed by several column chromatographies. The molecular mass of the purified enzyme was estimated to be 52.6 kDa by SDS-PAGE, although gel filtration analysis of the native enzyme suggests it exists as a homodimer in solution. The purified enzyme showed maximal activity at pH 5.0 and 70 °C. The enzyme was highly specific toward a fucosyl-monosaccharide (Fuc-α-pNP), but it also showed activity toward 2-sulfo-Fuc-α-pNP and fucosyl-α-lactosides (Fuc-α-Galβ1→4Glc-β-pNP). We determined the primary structure of the α- l -fucosidase and validated its expression level in starfish tissue. Whole genome sequence analysis of P. pectinifera was also performed in the present study. Detailed primary structural analysis using bioinformatics tools revealed Pap-Alf lacks the C-terminal region that is otherwise conserved in all previously described α- l -fucosidases. Quantitative gene expression analysis of Pap-Alf in each tissue indicated that the expression of Pap-Alf gene in pancreas was 5-fold higher than in ovary.

Fucose is an important component of many oligo- and polysaccharide structures as well as glycoproteins and glycolipids, which are often associated with a variety of physiological processes ranging from fertilization, embryogenesis, signal transduction, and disease progression, such as rheumatoid arthritis, inflammation, and cancer. The enzyme α-l-fucosidase is involved in the cleavage of the fucosidic bond in glycans and glycoconjugates, particularly the Fuc-α-1,2-Gal, Fuc-α-1,3/4-GlcNAc, and Fuc-α-1,6-GlcNAc linkages. Here, we report a highly efficient fucosidase, designated as BfFucH identified from a library of bacterial glycosidases expressed in E. coli from the CAZy database, which is capable of hydrolyzing the aforementioned fucosidic linkages, especially the α-1,6-linkage from the N-linked Fuc-α-1,6-GlcNAc residue on glycoproteins. Using BfFucH coupled with endoglycosidases and the emerging glycosynthases allows glycoengineering of IgG antibodies to provide homogeneous glycoforms with well-defined glycan structures and optimal effector functions.

Recently, we established two mouse monoclonal antibodies (R-10G and R-17F). The R-17F antibody (IgG1 subtype) exhibited a strong cytotoxic effect on hiPS/ES cells. The R-17F antigen isolated from a total lipid extract of hiPS (Tic) cells was identified as LNFP I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc). In the present study, R-17F binding proteins were isolated from hiPS (Tic) cell lysates with an affinity column of R-17F. They gave one major R-17F positive band around 250 kDa, and several minor bands between 150 kDa and 25 kDa. The former band was identified as podocalyxin by LC/MS/MS after SDS-PAGE. Hapten inhibition studies on R-17F binding to R-17F column-purified proteins with various synthetic oligosaccharides revealed that the blood group H type 1 triaose structure (Fucα1-2Galβ1-3GlcNAc) was the predominant epitope on all the R-17F binding proteins. These bands disappeared completely on digestion with α1-2 fucosidase, but not with α1-3/4 fucosidase. Upon PNGase F digestion, the R-17F positive band around and above 250 kDa did not show any change, while the minor bands between 150 kDa and 25 kDa disappeared completely, suggesting that the epitope is expressed on N-glycans in the latter and probably on O-glycans in the former. These results, together with those obtained in our previous studies on R-10G (Kawabe et al. Glycobiology, 23, 322-336 (2013)), indicated that both R-10G and R-17F epitopes are carried on the same podocalyxin molecule. The R-17F epitopes on these glycoproteins expressed on hiPS cells could be associated with the molecular mechanism underlying the carbohydrate-mediated cytotoxic activity of R-17F.

Three novel bacterial α-l-fucosidases, which cleave terminal fucosyl residues from glycoconjugates are reported in this work. Originating from the recently discovered bacterium Emticicia oligotrophica, recombinant fucosidase isoforms designated as Eo0918, Eo3066 and Eo3812 were shown to have the highest activity between pH 6.0 and 7.0 and temperature optima between 30 and 45°C. All enzymes catalyzed the hydrolysis of the model substrate pNP-α-l-fucose and revealed significantly different regiospecificities towards fucose-containing oligosaccharides: Eo0918 liberated exclusively α1,6-linked fucose and Eo3812 released only α1,3-fucosyl residues, whereas Eo3066 showed broader substrate promiscuity. The enzymatic activity of Eo0918 and Eo3812 increased upon the addition of Ca(2+), Mn(2+) and Zn(2+) ions, whereas the activity of Eo3066 was significantly decreased in the presence of these metal ions. In addition, Eo0918 also catalyzed the transfer of fucose from pNP-α-l-fucose to the 7-hydroxyl group of 4-methylumbelliferone with up to 15% transglycosylation yield. Facile recombinant expression in E. coli, distinct substrate specificities and the transglycosylation ability of Eo0918 presented herein make these newly discovered fucosidases valuable candidates for bioanalytical and biotechnological applications.

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.

α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

The α-1,3/4-l-fucosidases (EC 3.2.1.111; GH29) BbAfcB from Bifidobacterium bifidum and CpAfc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto-N-fucopentaose II (LNFP II) through regioselective transfucosylation of lacto-N-tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering BbAfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of BbAfcB with the corresponding 17-aminoacid α-helical loop of CpAfc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT BbAfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4-l-fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ± 2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT BbAfcB (14 ± 0.3%) and comparable to that obtained with CpAfc2 (50 ± 8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto-N-neotetraose (LNnT) could be enriched with LNFP III.

Sperm–oocyte interaction during fertilization is multiphasic, with multicomponent events, taking place between egg's glycoproteins and sperm surface receptors. Protein–carbohydrate complementarities in gamete recognition have observed in cases throughout the whole evolutionary scale. Sperm-associated α- l -fucosidases have been identified in various organisms. Their wide distribution and known properties reflect the hypothesis that fucose and α- l -fucosidases have fundamental function(s) during gamete interactions. An α- l -fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven species across the genus Drosophila . Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster . In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α- l -fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. Our results showed that α- l -fucosidase bound to fucose residue and in particular it prefers N -glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N -glycans carrying only a terminal mannose residue. The ability of sperm α- l -fucosidase to bind to the micropylar chorion and to the vitelline envelope was examined in in vitro assays in presence of α- l -fucosidase, either alone or in combination with molecules containing fucose residues. No binding was detected when α- l -fucosidase was pre-incubated with fucoidan, a polymer of α- l -fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α- l -fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm–egg interactions in Drosophila .

α-L-Fucosidase activity is associated with several diseases. To study the enzymatic activity change under pathological conditions, we developed a quinone methide-generating activity-based probe useful for examining the presence, activity, and localization of human α-L-fucosidase in vivo in the context of Helicobacter pylori infection. In particular, an increase in intracellular fucosidase (Fuca1) activity was found in gastric epithelial cells upon bacterial infection. We further studied the effect of several bacterial stimulants on this enhanced Fuca1 activity and identified lipopolysaccharides to be a major contributing factor.

Fucosidase inhibitors represent captivating targets for various biological applications. Iminosugars featuring a fluoroalkyl chain in the pseudoanomeric position were synthesized by nucleophilic addition of a perfluoroalkyl Grignard reagent to a nitrone. Reduction of the N–O bond of the hydroxylamine was achieved by treatment with an aqueous solution of sulfur dioxide. The pKa = 4.5 of the target pyrrolidine reflected a strong electronic effect of the fluoroalkyl chain. Inhibitory potencies of the fluorinated iminosugars and their corresponding hydroxylamines were evaluated against α-L-fucosidase at various pH values.

Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multi-step chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core-fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide, but also to high-mannose and complex type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

The presence and activity of glycosidases in Irish bovine milk over three months of lactation was investigated. A low level of variation between animals was observed and the highest level of glycosidase activity was present in colostrum, decreasing through transitional milk production to minimal but constant levels in mature milk. N-acetyl-β- d -glucosaminidase, α- l -fucosidase, α-galactosidase and N-acetyl-neuraminidase appear to be the most biologically relevant glycosidases in bovine milk. The elevated levels of enzymatic activity in colostrum suggests the milk glycosidases may play a role in the digestion of bovine milk glycans in the infant mammal, possibly acting as substitutes for bacterial glycosidases prior to colonisation by the gut microflora, which are involved in in vivo oligosaccharide metabolism in the lower gastrointestinal tract.

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite −1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite −1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

We present a study of the thermal inactivation and variation of activity of two enzymes in sheep milk: γ-glutamyltransferase and α- l -fucosidase. We find that the thermal treatment of milk causes inactivation of α- l -fucosidase between 57 °C and 68 °C, and γ-glutamyltransferase between 68 °C and 80 °C. In untreated sheep milk, the highest level of enzymatic activity (EA) for these two enzymes is in the pH range 5.5–6.0 and 8.0–8.5, respectively. Skimming treatment causes the loss of 35% of the activity of γ-glutamyltransferase. No differences in the EA were ascribable to the diets evaluated in this study. We observed EAs of α- l -fucosidase and γ-glutamyltransferase over the entire lactation cycle, noting that the EA of the former increased from 81 to 207 U mL−1 (average 159 ± 30 U mL−1) while the EA of the latter from 3.12 to 4.89 U mL−1 (average 3.83 ± 0.34 U mL−1). Relationships between both enzymes and selected milk parameters (lipids, proteins, lactose, urea and somatic cells) are highlighted by Principal Component Analysis.

Background The synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases. Methods The issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated. Results Both FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase. Conclusions The sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases. General significance The action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases.

To probe the utility of a multivalent approach for fucosidase inhibition, a series of di- and tri-valent imino sugars based on L-fuco-configured 1,4-imino- and 1,4-bis(imino)-cyclitol epitopes has been synthesized and analyzed for fucosidase inhibition with the best trivalent species yielding a modest improvement in binding constant. Structural analysis of a representative pair of mono- and tri-valent imino sugars has been performed on a bacterial fucosidase, BtFuc2970. The 3D structures show binding of the imino-cyclitol in the 3E conformation, consistent with the known pathway for fucosidase action.

The mirror image of natural product (+)-adenophorine along with its 1-epi-, 1-homo-analogs and other derivatives have been synthesized and evaluated as glycosidase inhibitors. The synthetic strategy is based on the skeletal rearrangement of tetrahydroxylated C -alkyl azepanes obtained via a Staudinger/azaWittig/alkylation sequence starting from a sugar-derived azidolactol. Several organometallic species have been investigated for the alkylation step including organomagnesium, organolithium, organozinc, organoaluminum and organocerium reagents. While diallylzinc proved to be the most efficient to introduce an allyl substituent, disappointing results were obtained with the other organometallic species leading either to lower yields or no reaction. Enzymatic assays indicate that (−)-adenophorine is a moderate α- l -fucosidase inhibitor.

A thermally induced intramolecular 1,3-dipolar cycloaddition of an azidoester and subsequent sodium cyanoborohydride reduction of the resulting bicyclic vinylogous urethane to give a bicyclic aminolactone allows access to an eight carbon homologue of α-homomannojirimycin which is a weak fucosidase inhibitor. Intermediates with both an α- and β-amino acid moiety are described and may be useful for incorporation of homopipecolic acids into novel peptide libraries.

Many polyhydroxylated alkaloids from plants are specific inhibitors of glycosidases. Information about these has led to the development of a wide range of both naturally occurring and synthetic inhibitors which may be used for mechanistic studies and for the purification of these enzymes. Sugar lactones are starting materials for highly efficient syntheses of deoxymannojirimycin and deoxyfuconojirimycin and of a number of their iminoheptitol analogues. This has allowed an investigation of the relationship between mannosidase and fucosidase inhibition.

α-l-Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α-l-Fucosidases, glycoside hydrolases for catalyzing the removal of α-l-fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α-l-fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α-l-fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α-l-fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α-l-fucosidases.

Here, we report the biochemical characterization of a novel α- l -fucosidase with broad substrate specificity (FpFucA) isolated from the mycelial fungus Fusarium proliferatum LE1. Highly purified α- l -fucosidase was obtained from several chromatographic steps after growth in the presence of l -fucose. The purified α- l -fucosidase appeared to be a monomeric protein of 67 ± 1 kDa that was able to hydrolyze the synthetic substrate p -nitrophenyl α- l -fucopyranoside ( p NPFuc), with K m = 1.1 ± 0.1 mM and k cat = 39.8 ± 1.8 s −1 . l -fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited p NPFuc hydrolysis, with inhibition constants of 0.2 ± 0.05 mM, 7.1 ± 0.05 nM, and 12.2 ± 0.1 mM, respectively. We assumed that the enzyme belongs to subfamily A of the GH29 family (CAZy database) based on its ability to hydrolyze practically all fucose-containing oligosaccharides used in the study and the phylogenetic analysis. We found that this enzyme was a unique α- l -fucosidase that preferentially hydrolyzes the α-(1 → 4)- L -fucosidic linkage present in α- L -fucobiosides with different types of linkages. As a retaining glycosidase, FpFucA is capable of catalyzing the transglycosylation reaction with alcohols (methanol, ethanol, and 1-propanol) and p NP-containing monosaccharides as acceptors. These features make the enzyme an important tool that can be used in the various modifications of valuable fucose-containing compounds.

International audience; A small library of divalent fucosidase inhibitors containing pyrrolidine motifs and separated by polyamino and triazole-benzylated spacers was prepared and evaluated as α-fucosidase inhibitors. Although a weak multivalent effect was observed in polyamino derived dimers, useful structural information can be deduced about the length of the bridge, the number of nitrogen atoms present and the moieties close to the pyrrolidine. Within these investigations one of the best α-fucosidase inhibitors containing a pyrrolidine framework was obtained (18, Ki = 3.7 nM).

International audience; The synthesis and the biological evaluation of a new ferrocenyl-iminosugar conjugate designed for fucosidase inhibitory and anticancer activity is described. The compound showed strong affinity for fucosidase from bovine kidney (Ki = 23 nM) and from Bacteroides thetaiotaomicron (Ki = 150 nM), displaying a 10-fold tighter binding affinity for these enzymes than the previous analogs. The interaction pattern that improves binding has been evaluated through structural analysis of the inhibitor–enzyme complex. The ferrocenyl-iminosugar exhibits significant anticancer activity on MDA-MB-231 and SK-MEL28 cell lines at 100 μM.

International audience; Multi-valent inhibitors offer promise for the enhancement of therapeutic compounds across a range of chemical and biological processes. Here, a significant increase in enzyme-inhibition potencies was observed with a dimeric iminosugar-templated fucosidase inhibitor (IC50 = 0.108 μM) when compared to its monovalent equivalent (IC50 = 2.0 μM). Such a gain in binding is often attributed to a “multivalent effect” rising from alternative recapture of the scaffolded binding epitopes. The use of control molecules such as the meso analogue (IC50 = 0.365 μM) or the enantiomer (IC50 = 569 μM), as well as structural analysis of the fucosidase-inhibitor complex, allowed a detailed analysis of the possible mechanism of action, at the molecular level. Here, the enhanced binding affinity of the dimer over the monomer can be attributed to additional interactions in non-catalytic sites as also revealed in the 3-D structure of a bacterial fucosidase inhibitor complex.

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.

The nitrone 4 (4,5-dideoxy-4-hydroxylamino-3,4-O-isopropylidene-L-lyxofuranose) was synthesised from D-ribose and used as key intermediate for the preparation of fucosidase inhibitors. We describe two transformations of 4. Hydrolysis with aqueous sulfur dioxide gave the known potent nanomolar inhibitor 4-amino-4,5-dideoxy-L-lyxofuranose (3). 1,3-Dipolar cycloaddition with enol ethers led to the related 1,2,5,6-tetradeoxy-2,5-imino-L-altroheptonic ester 2a, acid 2b and the corresponding heptitol 2c. The new iminosugars have been evaluated for their inhibitory activity against α-L-fucosidase from bovine kidney. The alcohol 2c turned out to be a potent inhibitor in the same range as the amino-sugar 3 (K(i)=8 vs 10nM).

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 μM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

The synthesis of a series of aryl-substituted cyclic sugar imines was performed via a tandem nucleophilic addition/substitution reaction. The so-obtained ketimines displayed fucosidase inhibitory activities (IC 50 = 46–556 μM). Their reduced counterparts were prepared and assayed after addition of sodium borohydride to the enzymatic assay stock solution. The pyrrolidines strongly inhibit fucosidase (IC 50 = 0.65–150 μM).

The glycosidase activities (beta-D-mannosidase, alpha-L-fucosidase and N-acetyl-beta-D-glucosaminidase) have been compared in whole and split ejaculate samples from men whose couple suffers from infertility. The site of secretion of enzymatic activities is either prostatic or epididymal. The three enzymatic activities have possibly different origin and should constitute new biochemical markers of male genital tract.

The inhibitory activities against glycosidases of synthetic thiasugars, the ring sulfur analogs of carbohydrate, were surveyed with a special emphasis on our own studies. 5-Thio-L-fucose, the ring sulfur analog of L-fucose, was the first thiopyran that shows a K(i) value in the micromolar range against a glycosidase. The structure-activity relationship studies for the fucosidase inhibition by 5-thio-L-fucose disclosed that a hydrophobic interaction between the ring sulfur atom and the enzyme is responsible for the strong binding. The syntheses and activities of di- and trisaccharide analogs incorporating thiasugars are also outlined. These oligosaccharide analogs are glycosidase-resistant and some of them show strong binding to antibodies or lectins. We created a sulfylimine compound having a thiafuran structure as a transition-state analog inhibitor of glucosidases and found that it has a weak inhibition against a glucosidase. The same thiafuran structure could be found in a natural product, salacinol, which was isolated and elucidated to be a strong glucosidase inhibitor by another group. A study of the structure-activity relationship using the synthetic analogs of salacinol indicated the relevance of both the sulfonium ion in the thiafuran ring and its intramolecular counter anion, sulfate, to the inhibition activity.