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The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 μM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase. [ABSTRACT FROM AUTHOR]

Enzymes capable of modifying the sulfated polymeric molecule of fucoidan are mainly produced by different groups of marine organisms: invertebrates, bacteria, and also some fungi. We have discovered and identified a new strain of filamentous fungus Fusarium proliferatum LE1 (deposition number in Russian Collection of Agricultural Microorganisms is RCAM02409), which is a potential producer of fucoidan-degrading enzymes. The strain LE1 (RCAM02409) was identified on the basis of morphological characteristics and analysis of ITS sequences of ribosomal DNA. During submerged cultivation of F. proliferatum LE1 in the nutrient medium containing natural fucoidan sources (the mixture of brown algae Laminaria digitata and Fucus vesiculosus), enzymic activities of α- L-fucosidase and arylsulfatase were inducible. These enzymes hydrolyzed model substrates, para-nitrophenyl α- L-fucopyranoside and para-nitrophenyl sulfate, respectively. However, the α- L-fucosidase is appeared to be a secreted enzyme while the arylsulfatase was an intracellular one. No detectable fucoidanase activity was found during F. proliferatum LE1 growth in submerged culture or in a static one. Comparative screening for fucoidanase/arylsulfatase/α- L-fucosidase activities among several related Fusarium strains showed a uniqueness of F. proliferatum LE1 to produce arylsulfatase and α- L-fucosidase enzymes. Apart them, the strain was shown to produce other glycoside hydrolyses. [ABSTRACT FROM AUTHOR]

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 p Ka = 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. [ABSTRACT FROM AUTHOR]

Schistosoma mansoni is one of the major agents of the disease Schistosomiasis, which is one of the major global public health concerns. Biomphalaria glabrata is an obligate intermediate mollusc host of S. mansoni . Although the development of S. mansoni occurs in the snail hepatopancreas, studies that focus on this organ remain limited. In this study, we biochemically identified five distinct carbohydrases (amylase, maltase, α-glucosidase, trehalase, and α-L-fucosidase), lipases, and peptidases in the B. glabrata hepatopancreas and focused on the isolation and characterization of the activity of α-L-fucosidase. The isolated α-L-fucosidase has a molecular mass of 141 kDa, an optimum pH of 5.8, and is inhibited by Tris, fucose, and 1-deoxyfuconojirimycin. B. glabrata α-L-fucosidase is an exoglycosidase that can hydrolyze the natural substrate fucoidan to fucose residues. It presented K m values of 48.4 μM to 4-Methylumbelliferyl α-L-fucopyranoside and 0.55 mM to p-nitrophenyl-α-L-fucopyranoside. Thus, α-L-fucosidase has a high activity in the hepatopancreas of B. glabrata , and the differential expression of this enzyme between susceptible and resistant strains indicates that besides its digestive role, α-L-fucosidase may also be important in host/parasite interactions. [ABSTRACT FROM AUTHOR]

The activity of α-L-fucosidase in oviductal fluid increases around the time of ovulation. α-L-fucosidase is also associated with the spermatozoal plasma membrane and its substrate, fucose, has been identified in the zona pellucida (ZP) and on the spermatozoal surface, suggesting a role in fertilisation. The aim of the present study was to investigate the role of exogenous α-L-fucosidase during fertilisation. Porcine oocytes were incubated with fucosidase and later subjected to in vitro fertilisation (1VF). No effect on the percentage of oocytes fertilised was observed, although there was a slight decrease in spermatozoa-ZP binding. Fucosidase was then added to IVF medium, and spermatozoa and oocytes were coincubated for 15 min. A significant increase in spermatozoa-ZP binding and penetration was observed, suggesting a role of the enzyme in the fertilisation ability of spermatozoa. In addition, fluorescence intensity and the patterns of spermatozoa membrane-associated α-L-fucosidase distribution, as assessed by indirect immunofluorescence, were not affected by the presence or absence of exogenous enzyme, suggesting an independent role for the exogenous and spermatozoa-associated enzymes. Addition of exogenous α-L-fucosidase increased the spermatozoal intracellular ionised calcium concentration and tyrosine phosphorylation, suggesting a role in promoting capacitation and, at the same time, protecting spermatozoa from a premature acrosome reaction. Thus, α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the diagnostic efficiency for hepatocellular carcinoma (HCC) with the combined analysis of alpha- l -fucosidase (AFU), alpha-fetoprotein (AFP) and thymidine kinase 1 (TK1). Serum levels of AFU, AFP and TK1 were measured in: 116 patients with HCC, 109 patients with benign hepatic diseases, and 104 normal subjects. The diagnostic value was analyzed using the logistic regression equation and receiver operating characteristic curves (ROC). Statistical distribution of the three tested tumor markers in every group was non-normally distributed (Kolmogorov–Sminov test, Z = 0.156–0.517, P < 0.001). The serum levels of AFP and TK1 in patients with HCC were significantly higher than those in patients with benign hepatic diseases (Mann–Whitney U test, Z = −8.570 to –5.943, all P < 0.001). However, there was no statistically significant difference of AFU between these two groups (Mann–Whitney U test, Z = −1.820, P = 0.069). The levels of AFU were significantly higher in patients with benign hepatic diseases than in normal subjects (Mann–Whitney U test, Z = −7.984, P < 0.001). Receiver operating characteristic curves (ROC) in patients with HCC versus those without HCC indicated the optimal cut-off value was 40.80 U/L for AFU, 10.86 μg/L for AFP and 1.92 pmol/L for TK1, respectively. The area under ROC curve (AUC) was 0.718 for AFU, 0.832 for AFP, 0.773 for TK1 and 0.900 for the combination of the three tumor markers. The combination resulted in a higher Youden index and a sensitivity of 85.3%. The combined detection of serum AFU, AFP and TK1 could play a complementary role in the diagnosis of HCC, and could significantly improve the sensitivity for the diagnosis of HCC. [ABSTRACT FROM AUTHOR]

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background:Preoperative alpha-L-fucosidase (AFU) has been used as a diagnostic biomarker for hepatocellular carcinoma (HCC), but its role as a prognostic predictor after partial hepatectomy has not been well defined. The study aimed to investigate the prognostic significance of preoperative serum AFU for HCC patients after hepatic resection.Methods:A retrospective training data set and a prospective validation data set were used to evaluate the prognosis of HCC after partial hepatectomy. A total of 669 patients with histopathologically confirmed HCC were enrolled. Univariate and multivariate analyses were used to identify the prognostic significance of preoperative serum AFU.Results:The retrospective training data set showed a preoperative AFU>35 u l−1 should be used. The prospective validation data set showed preoperative AFU was an independent prognostic factor of overall survival (OS) (P=0.008; hazard ratio: 2.333; 95% confidence interval: 1.249-4.369). Patients with a preoperative AFU>35 u l−1 had a lower recurrence-free survival rate and an OS rate than those with AFU35 u l−1, and they have a higher tendency to form macrovascular invasion. Furthermore, the prognostic significance of AFU>35 u l−1 could also be applied to patients with alpha-fetoprotein levels of 400 ng ml−1.Conclusions:Preoperative serum AFU is a prognostic predictor of HCC. [ABSTRACT FROM AUTHOR]

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 3 E conformation, consistent with the known pathway for fucosidase action. [ABSTRACT FROM AUTHOR]

Abstract: Synthesis and inhibitory activities against α-l-fucosidase of phenylalkyl-substituted polyhydroxypiperidines have been described. Among the newly synthesized compounds, 2,4,6-trichloro derivative (16q) showed very high inhibitory activity against α-l-fucosidase with IC50 value of 0.005 μM, and K i values of 0.0011 μM, respectively. [Copyright &y& Elsevier]

A review dealing with 1,4-iminoalditol (hydroxylated pyrrolidine) derivatives as inhibitors of ?-L-fucosidases including the different synthetic approaches for their preparation as well as their inhibitory properties is presented.

Abstract: In this study we revealed that the addition of an N-phenylacetamide substituent to the C-1 position of 1-deoxyfuconojirimycin (DFJ) can lead to highly potent inhibitors of α-l-fucosidases. A structure–activity relationship study showed that a fluoro group on the phenyl ring greatly increased its potency and selectivity. In contrast the addition of two or three fluoro groups decreased their inhibition potency. Consequently, N-(2-fluorophenyl)-2β-DFJ acetamide (18j) was found to display very potent and selective inhibition of bovine kidney, rat epididymis, and human lysosome α-l-fucosidases, with IC50 value of 0.012, 0.044, and 0.0079μM respectively. It is noteworthy that our designed N-phenyl-2β-DFJ acetamide derivative exhibited about 18-fold stronger effects on human lysosomal α-l-fucosidase than original DFJ and it occupied the active-site of this enzyme. We therefore expect that this compound may find applications in new therapeutic trials against genetic deficiency disorders. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOS) are not digested in the proximal intestine. In distal intestine, HMOS collectively modify the microbiota, but the response of individual bacteria to individual components of the HMOS is not well defined. Here, each of 25 major isolates of the human intestinal microbiota was fed individual major fucosylated and sialylated HMOS in anaerobic culture. This allowed for an assessment of the influence of specific HMOS on the growth and metabolic products of individual microbiota bacteria. Most Bifidobacteria spp. and Bacteroides spp. grew, induced α-l-fucosidase activity, and produced abundant lactate or short-chain fatty acids (SCFAs) when fed 2′-fucosyllactose (2′-FL), 3-FL, and lactodifucotetraose (LDFT). Lactobacillus delbrueckii ATCC7830, Enterococcus faecalis ATCC19433, and Streptococcus thermophilus ATCC19258 exhibited slight growth, pH reduction, and lactate production when supplemented with 2′-FL or 3-FL, but not LDFT. Supplementation with 3′-sialyllactose (3′-SL) and 6′-SL promoted moderate growth of Bifidobacterium longum JCM7007, 7009, 7010, 7011, 1272, 11347, ATCC15708, Bacteroides vulgatus ATCC8482, and B. thetaiotaomicron ATCC29148; accordingly, these bacteria exhibited greater neuraminidase activity and produced copious lactate, SCFA, or both. Lactobacillus delbrueckii ATCC7830 also consumed 6′-SL. In contrast, Clostridium spp., L. rhamnosus ATCC53103, E. faecalis ATCC29200, Staphylococcus spp., Enterobacter spp., and Escherichia coli K12 did not consume milk oligosaccharides nor produce appreciable acidic fermentation products. Specific Bifidobacteria and Bacteroides differentially digest specific individual HMOS, with the major fucosylated milk oligosaccharides most strongly stimulating key species of mutualist symbionts. This suggests strategies for treating dysbiosis of the microbiota and associated inflammatory disorders. [ABSTRACT FROM PUBLISHER]

Highlights: [•] Chitinase, fucosidase and glucosaminidase are active in A. cajennense midgut. [•] A. cajennense carbohydrases are acidic digestive enzymes. [•] It is the first time that α-l-fucosidase activity was isolated from tick midgut. [•] A. cajennense α-l-fucosidase hydrolyzes fucoidan. [•] α-l-fucosidase could be related to defense against infection. [ABSTRACT FROM AUTHOR]

Bifidobacteriumis a genus of Gram-positivebacteria, which is important in the absorption of nourishment fromthe human milk oligosaccharides (HMO). We present here the detailedsimulation of the enzymatic hydrolysis of 2′-fucosyllactosecatalyzed by 1,2-α-l-fucosidase from Bifidobacterium bifidumusing the combined quantummechanical and molecular mechanical approach. Molecular dynamics simulationsand free energy profiles support that the overall reaction is a stepwisemechanism. The first step is the proton transfer from N423 to D766,and the second step involves the hydrolysis reaction via the inversionmechanism catalyzed by the amide group of N423. Assisted by D766,N423 serves as the general base to activate the water molecule toattack the anomeric carbon center. E566 is the general acid to facilitatethe cleavage of glycosidic bond between l-fucose and galactoseunits. The intrinsic resonance structure for the side chain amidegroup of the asparagine residue is shown to be the origin to the catalyticactivity, which is also confirmed by the mutagenesis simulation ofN423G. [ABSTRACT FROM AUTHOR]

In this work, focused on possible application of α-l-fucosidases from bacterial sources in the synthesis of α-l-fucosylated glycoconjugates, several nonpathogenic aerobic bacterial strains were screened for α-l-fucosidase activity. Among them Paenibacillus thiaminolyticus was confirmed as a potent producer of enzyme with the ability to cleave the chromogenic substrate p-nitrophenyl α-l-fucopyranoside. The gene encoding α-l-fucosidase was found using the genomic library of P. thiaminolyticus constructed in the cells of Escherichia coli DH5α and sequenced (EMBL database: FN869117, carbohydrate-active enzymes database: Glycosidase family 29). The enzyme was expressed in the form of polyhistidine-tagged protein (51.2 kDa) in Escherichia coli BL21 (DE3) cells, purified using nickel-nitrilotriacetic acid agarose affinity chromatography and characterized using the chromogenic substrate p-nitrophenyl α-l-fucopyranoside (Km = (0.44 ± 0.02) mmol/L, KS = (83 ± 8) mmol/L (substrate inhibition), pHoptimum = 8.2, toptimum = 48°C). By testing the ability of the enzyme to catalyze the transfer of α-l-fucosyl moiety to different types of acceptor molecules, it was confirmed that the enzyme is able to catalyze the formation of α-l-fucosylated p-nitrophenyl glycopyranosides containing α-d-galactopyranosidic, α-d-glucopyranosidic, α-d-mannopyranosidic or α-l-fucopyranosidic moiety. This enzyme is also able to catalyze α-l-fucosylation of aliphatic alcohols of different lenghs of alkyl chain and hydroxyl group positions (methanol, ethanol, 1-propanol, 2-propanol and 1-octanol) and hydroxyl group-containing amino acid derivatives (N-(tert-butoxycarbonyl)-l-serine methyl ester and N-(tert-butoxycarbonyl)-l-threonine methyl ester). These results indicate the possibility of exploiting this enzyme in the synthesis of different types of α-l-fucosylated molecules representing compounds with potential application in biotechnology and the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

While the catalytic nucleophile in the configuration-retaining α-L-fucosidases from family GH29 is fully conserved with respect to sequence, there is no fully sequence-conserved acid/base residue candidate across the family. X-ray crystallographic studies and kinetic characterizations have allowed the identification of this residue in a few cases, and a recent combination of phylogenetic tree analyses with substrate specificity data has allowed the division of GH29 enzymes into two subfamilies, A and B, allowing the probable assignment of these residues. Here, we perform detailed kinetic and mechanistic characterizations of the corresponding alanine mutants and other candidates. Through comparison of kinetic parameters obtained for the hydrolysis of fucosyl substrates with activated leaving groups by these mutants with those of the corresponding wild-type enzymes, in conjunction with the demonstration of azide rescue, we largely confirm the acid/base residue predictions for the GH29 fucosidases from the two subfamilies. [ABSTRACT FROM AUTHOR]

Abstract: Fucosidases, enzymes that cleave fucose from the non-reducing end of a glycan, represent promising medicinal targets reflecting their roles in cancer metastasis, inflammation, host-parasite interactions and the lysosomal storage disorder fucosidosis. The X-ray crystal structures of Bacteroides thetaiotaomicron GH29 α-l-fucosidase (BtFuc2970) in a new crystal form (at a resolution of 1.59Å) and liganded with a 5-membered iminocyclitol inhibitor (1.73Å) are reported herein. The 5-membered iminocyclitol binds in a 3E conformation, mimicking the proposed 3H4 half chair transition-state of the enzyme catalysed reaction, and its K i for BtFuc2970 was determined as 2μM. Structural analysis of fucosidase inhibition through 5-membered iminocyclitols will aid in the rational design of more potent fucosidase inhibitors for treatment of a range of medical conditions. [Copyright &y& Elsevier]

In previous studies we described a decreased alpha-L-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-L-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-L-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-L-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-L-fucosidase could be regulated at transcriptional level in colorectal cancer. [ABSTRACT FROM AUTHOR]

Enhanced metabolism of fucose through fucosidase overexpression is a signature of some cancer types, thus suggesting that fucosidase-targetted ligands could play the role of drug-delivery vectors. Herein, we describe the synthesis of a new series of pyrrolidine-ferrocene conjugates, consisting of a L- fuco-configured dihydroxypyrrolidine as the fucosidase ligand armed with a cytotoxic ferrocenylamine moeity. Three-dimensional structures of several of these fucosidase inhibitors reveal transition-state-mimicking 3 E conformations. Elaboration with the ferrocenyl moiety results in sub-micromolar inhibitors of both bovine and bacterial fucosidases, with the 3D structure of the latter revealing electron density indicative of highly mobile alkylferrocene compounds. The best compounds show a strong antiproliferative effect, with up to 100 % inhibition of the proliferation of MDA-MB-231 cancer cells at 50 μ M. [ABSTRACT FROM AUTHOR]

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on l-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene ( FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ∼1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily. [ABSTRACT FROM AUTHOR]

AlfB and AlfC α-L-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3-N-acetylglucosamine and fucosyl-α-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-α-L-fucopyranoside into fucosyl-α-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6-N-acetylglucosamine. [ABSTRACT FROM AUTHOR]

Highlights: [•] We synthesized 3-O-α-l-fucopyranosyl-d-mannopyranose by α-fucosidase. [•] 6I,6IV-Di-O-[α-l-Fuc-(1→3)-α-d-Man]-βCD} was chemically synthesized. [•] Fucosyl βCDs were investigated cell toxicity to colon cancer cell line, HCT116. [•] Fucosyl βCDs are expected useful as drug carriers in drug delivery systems. [Copyright &y& Elsevier]

Background: Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds. Results: A bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and ß-linked oligosaccharides, and had remarkably high ß- galactosidase, ß-glucosidase and ß-fucosidase activities. Because of the lack of information about the usefulness of ß-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for ß-glucosidase or ß-galactosidase. The BglMKg had maximal ß-galactosidase and ß-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for ß-galactosidase activity was 6.5, whereas the optimum pH for ß-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed ß- glucosidase substrates than ß-galactosidase ones. Conclusions: BglMKg is a small, monomeric, cold-active ß-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications. [ABSTRACT FROM AUTHOR]

Abstract: The ability of a series of pyrrolidines to inhibit several glycosidases was investigated. Using Fleet’s ‘mirror-image postulate’, it was proposed that enantiomeric derivatives of 1,4-dideoxy-1,4-imino-d-lyxitol (a known α-d-galactosidase inhibitor) would show inhibitory activity against α-l-fucosidases. Some modest α-l-fucosidase inhibitory activity was observed for selected compounds (particularly an aminomethyl pyrrolidine) and it was proposed that better activity could be obtained by modifying the C-2 side chain of the pyrrolidine core. The d-galacto carbamate scaffold also exhibited somewhat selective, albeit modest, α-l-fucosidase inhibitory activity and may prove to be an interesting scaffold for further development. [Copyright &y& Elsevier]

Abstract: Four branched tetra- and pentahydroxylated azepanes have been synthesized from a common azepane precursor through dihydroxylation followed by deoxygenation. They have been assayed as glycosidase inhibitors on a panel of 22 glycosidases and one methylated azepane displayed selective, competitive, and moderate inhibition toward bovine kidney α-l-fucosidase. [Copyright &y& Elsevier]

We have previously characterized from Lactobacillus casei BL23 three a-L-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3- N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the L-fucose moiety during growth on Fuc-a-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the a-L-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-a-l,3-GlcNAc. This proves that AlfB is involved in Fuc-a-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-a-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an a-L-fucosidase in lactic acid bacteria and the utilization of Fuc-a-1,3-GlcNAc as a carbon source for bacteria. [ABSTRACT FROM AUTHOR]

Lyme disease (LD) is the most prevalent tick-borne disease in Europe. LD is caused by the spirochete Borrelia burgdorferi. LD is a chronic disease which can attack a number of organs: skin, heart, brain, joints. Chronic, low-grade inflammation involves general production of pro-inflammatory cytokines and inflammatory markers and is a typical feature of aging. So far, the best method of diagnosing LD is a time-consuming and expensive two-stage serological method. The aim of our study was to evaluate the activity of two lysosomal exoglycosidases: α-fucosidase (FUC) and β-galactosidase (GAL) in the serum of patients with Lyme disease, as potential markers of LD. Due to the increasing number of patients with Lyme disease and a number of false results, new ways to diagnose this disease are still being sought. As elevated level of β-galactosidase is a manifestation of residual lysosomal activity in senescent cells, the increase in its activity in serum during chronic Lyme disease might be a marker of a potentially accelerated senescence process. The study was performed on serum taken from cubital veins of 15 patients with Lyme disease and eight healthy subjects (control group). FUC and GAL activity was measured by the method of Chatterjee et al. as modified by Zwierz et al. In the serum of patients with Lyme disease, GAL activity significantly increased (p = 0.029), and the activity of FUC had a tendency to increase (p = 0.153), compared to the control group. A significant increase in GAL activity in the serum of patients with Lyme disease indicates an increased catabolism of glycoconjugates (glycoproteins, glycolipids, proteoglycans) and could be helpful in the diagnosis of Lyme disease, although this requires confirmation in a larger group of patients. As GAL is the most widely used assay for detection of senescent cells, an elevated level of β-galactosidase might be a manifestation of accelerated senescence process in the course of Lyme disease. [ABSTRACT FROM AUTHOR]

α-l-Fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-l-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-l-fucosynthase that specifically introduces Lea and Lex antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1–3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-l-fucosidase (EC 3.2.1.111) and α-l-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Lea/x epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds. [ABSTRACT FROM AUTHOR]

Abstract: In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC–MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates. [Copyright &y& Elsevier]

The enzyme α-l-fucosidase (AFu) plays an important role in the diagnosis of hepatocellular carcinoma (HCC) and fucosidosis. In this paper, a simple, sensitive and precise method based upon measuring the fluorescence quenching of CdTe semiconductor quantum dots (QDs) was developed for detecting the enzymatic activity of AFu. The detection limit of AFu was 0.01 U/L (n = 3) and the linear relationship was 0.01-4 U/L. The selectivity experiment indicated excellent selectivity for AFu over a number of interfering species. We have also studied the detection mechanism of AFu by X-ray photoelectron spectroscopy (XPS) and found that the quenching effect was caused by the oxidation of tellurium by 2-chloro-4-nitrophenol (2-CNP) which produced in AFu catalytic reaction. Moreover, the AFu sensor based on QDs was used satisfactorily for the assessment of AFu activity in serum samples. It will most probably be applicable in assembling diagnostic microdevice to realize the rapid clinic analysis of AFu. [ABSTRACT FROM AUTHOR]

Abstract: Background and study aims: Alphafoetoprotein (AFP) is not secreted in all cases of hepatocellular carcinoma (HCC) and may be normal in as many as 40% of patients with early HCC. Alpha-l-fucosidase (AFU), a lysosomal enzyme present in all mammalian cells, has been proposed as a tumour marker since many studies reported increased AFU serum levels in patients with cirrhosis and HCC. This study aimed at evaluation of the diagnostic value of AFU as a tumour marker of HCC and its prognostic role in follow-up after intervention. Patients and methods: This prospective case-control study was conducted on 80 patients (40 patients with HCC and 40 patients with chronic liver disease (CLD) as diseased controls) in addition to 40 apparently healthy individuals who served as a healthy control group. AFU serum levels in the three groups were measured and compared. Results: There was a statistically highly significant elevation (p <0.001) in the median values of serum AFU in the HCC group (0.99–26.59μmoll−1 min−1) when compared with both the CLD (0.11–4.60μmoll−1 min−1) and control (0.1–1.2μmoll−1 min−1) groups. At a cut-off value of 2.3005μmoll−1 min−1, AFU yielded a sensitivity and specificity of 90% and 97.5%, respectively. In HCC patients, AFU dropped significantly after successful intervention and can be used to follow-up therapy in these patients. Conclusion: AFU is a promising tumour marker in the diagnosis of HCC. It can be used also to follow up patients after interventional therapies. [Copyright &y& Elsevier]

The synthesis of 3-substituted L-fuco-azafagomines from D-lyxose is reported. They represent the first example of aza-C-glycosides having a biimino (-NH-NH-) moiety. The key step of the synthesis is the introduction of the hydrazine moiety by reductive hydrazination of a 1-deoxy-ketohexose with tert-butyl carbazate. Their glycosidase inhibitory properties are also reported. [ABSTRACT FROM AUTHOR]

Fucosylated carbohydrates and glycoproteins from human breast milk are essential for the development of the gut microbiota in early life because they are selectively metabolized by bifidobacteria. In this regard, α-L-fucosidases play a key role in this successful bifidobacterial colonization allowing the utilization of these substrates. Although a considerable number of α-L-fucosidases from bifidobacteria have been identified by computational analysis, only a few of them have been characterized. Hitherto, α-L-fucosidases are classified into three families: GH29, GH95, and GH151, based on their catalytic structure. However, bifidobacterial α-L-fucosidases belonging to a particular family show significant differences in their sequence. Because this fact could underlie distinct phylogenetic evolution, here extensive similarity searches and comparative analyses of the bifidobacterial α-L-fucosidases identified were carried out with the assistance of previous physicochemical studies available. This work reveals four and two paralogue bifidobacterial fucosidase groups within GH29 and GH95 families, respectively. Moreover, Bifidobacterium longum subsp. infantis species exhibited the greatest number of phylogenetic lineages in their fucosidases clustered in every family: GH29, GH95, and GH151. Since α-L-fucosidases phylogenetically descended from other glycosyl hydrolase families, we hypothesized that they could exhibit additional glycosidase activities other than fucosidase, raising the possibility of their application to transfucosylate substrates other than lactose in order to synthesis novel prebiotics. [ABSTRACT FROM AUTHOR]

A glycosidase enzyme with both β-glucosidase and β-fucosidase activities has been purified from the seeds of Dalbergia cochinchinensis Pierre (Thai Rosewood) by ammonium sulfate fractionation, preparative isoelectric focusing, and Sephadex G-150 chro-matography. The enzyme has molecular weights of 330,000 in the native state and 66,000 in the denatured state. Hydrolysis of p-NP-β-D-glucoside and p-NP-β-D-fucoside showed pH optimum at pH 5.0 and was inhibited by Rcat-gluconolactone, HgCl2, and p-chloromercuri-benzoate. The Km and fecal values of the purified enzyme were 5.4 mM and 307 s1 for p-NP-β-D-glucoside and 0.54 mM and 151 s−1 for p-NP-β-D-fucoside, so that the latter had by far the higher kcat&Km ratio. p-NP-β-D-galactoside, p-NP-β-D-xyloside, and p-NP-α-L-arabinoside were hydrolyzed more slowly. Hydrolysis of sophorose, laminaribiose, and gentiobiose were also rather slow, and hydrolysis of cellobiose was even slower. No hydrolysis of the cyanogenic glucosides linamarin or prunasin, but some hydrolysis of amygdalin and salicin was found. Further studies are required to identify the natural substrates of the enzyme. However, high yields, ease of purification, and storage stability of the enzyme make it a useful candidate for various applications, such as study of oligosaccharide synthesis by reversal of hydrolysis [ABSTRACT FROM AUTHOR]

Background: Alpha-fucosidase (FUC) is a lysosomal exoglycosidase involved in degradation of fucose-containing oligosaccharides, glycoproteins and glycolipids. Increased activities of FUC were earlier reported in the sera of alcohol dependent patients with alcoholic cirrhosis. The objective of this study was to determine serum and urinary activity of a-L-fucosidase after acute ethanol intoxication (binge drinking). Material/Methods: Activity of a-fucosidase was determined in serum and urine samples of eight healthy binge drinkers collected before acute ethanol ingestion (120―160 g) as well as 2, and 5 days thereafter using a colorimetric method. Results: After a binge drinking session, activity of FUC in the serum did not significantly increase, whereas urinary activity of FUC increased significantly on the fifth day after drinking. We found a positive correlation between serum FUC and serum aspartate aminotransferase levels measured on the 5th day after drinking and an inverse correlation between urinary FUC and serum alanine aminotransferase concentrations on a 2nd day after drinking. The cut-off value of 60 pKat/ml for urinary FUC activity demonstrated good sensitivity (87.5%) and specificity (87.5%). Conclusions: Binge drinking-induced increase in urinary activity of FUC is associated with liver dysfunction during alcohol consumption and can be detectable even five days thereafter. [ABSTRACT FROM AUTHOR]

The article discusses differences in the substrate specificities and active site structure of two-α-L-fucosidases enzymes present in Bacteroides thetaiotaomicron. It reports that α-L-fucosidases enzyme belongs to hydrolase family and is divided into two subfamilies based on phylogenetic clustering. It mentions that differences in structure of the enzyme were examined through analysis of various substrates at fixed concentration.

The freshwater snail Pomacea canaliculata , an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity, and origin of glycoside hydrolases along the digestive tract of P. canaliculata . A range of cellulases, hemicellulases, amylases, maltases, fucosidases, and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed among 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.

The sugar fucose plays a myriad of roles in biological recognition. Enzymes hydrolyzing fucose from glycoconjugates, α-l-fucosidases, are important targets for inhibitor and probe development. Here we describe the synthesis and evaluation of novel α-l-fucosidase inhibitors, with X-ray crystallographic analysis using an α-l-fucosidase from Bacteroides thetaiotamicron helping to lay a foundation for future development of inhibitors for this important enzyme class., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

A diagram is presented which explains the chemical process of Perfluoroalkylation of Nitrones for the Synthesis of a Series of Fucosidase Inhibitors.

Key intermediate (III) is converted into homologues (V) and (VIII) either by Lemieux-Johnson oxidation and subsequent Wittig reaction or in one step by Grubb metathesis, respectively. [ABSTRACT FROM AUTHOR]