β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

• Klíčová slova
• Pichia pastoris, Talaromyces flavus, site-directed mutagenesis, site-saturation mutagenesis, substrate specificity, β-N-acetylhexosaminidase,
• MeSH
• acetylgalaktosamin metabolismus   MeSH
• acetylglukosamin * metabolismus   MeSH
• acetylglukosaminidasa MeSH
• beta-N-acetylhexosaminidasy * metabolismus   MeSH
• kinetika MeSH
• mutace MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylgalaktosamin MeSH
• acetylglukosamin * MeSH
• acetylglukosaminidasa MeSH
• beta-N-acetylhexosaminidasy * MeSH

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.

• Klíčová slova
• enzymatic synthesis, glycosidase, human milk oligosaccharide, mutagenesis,
• MeSH
• Bifidobacterium bifidum * metabolismus   MeSH
• glykosidhydrolasy metabolismus   MeSH
• glykosyltransferasy metabolismus   MeSH
• lidé MeSH
• mateřské mléko * metabolismus   MeSH
• novorozenec MeSH
• oligosacharidy chemie   MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glykosidhydrolasy MeSH
• glykosyltransferasy MeSH
• oligosacharidy MeSH

Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

• Klíčová slova
• Glide docking, Talaromyces flavus, muramic acid, non-reducing carbohydrate, substrate specificity, transglycosylation, β-N-acetylhexosaminidases,
• MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• glykosidy metabolismus   MeSH
• glykosylace MeSH
• kinetika MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• oligosacharidy metabolismus   MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH
• glykosidy MeSH
• oligosacharidy MeSH

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.

• Klíčová slova
• galectin-3, molecular modeling, site-directed mutagenesis, solvent, substrate specificity, transglycosidase, β-N-acetylhexosaminidase,
• MeSH
• aktivace enzymů MeSH
• beta-N-acetylhexosaminidasy chemie   genetika   metabolismus   MeSH
• hydrolýza MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• mutace MeSH
• polysacharidy biosyntéza   chemie   farmakologie   MeSH
• proteinové inženýrství MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH
• polysacharidy MeSH

BACKGROUND: Galectin-3 (Gal-3) is a promising target in cancer therapy with a high therapeutic potential due to its abundant localization within the tumor tissue and its involvement in tumor development and proliferation. Potential clinical application of Gal-3-targeted inhibitors is often complicated by their insufficient selectivity or low biocompatibility. Nanomaterials based on N-(2-hydroxypropyl)methacrylamide (HPMA) nanocarrier are attractive for in vivo application due to their good water solubility and lack of toxicity and immunogenicity. Their conjugation with tailored carbohydrate ligands can yield specific glyconanomaterials applicable for targeting biomedicinally relevant lectins like Gal-3. RESULTS: In the present study we describe the synthesis and the structure-affinity relationship study of novel Gal-3-targeted glyconanomaterials, based on hydrophilic HPMA nanocarriers. HPMA nanocarriers decorated with varying amounts of Gal-3 specific epitope GalNAcβ1,4GlcNAc (LacdiNAc) were analyzed in a competitive ELISA-type assay and their binding kinetics was described by surface plasmon resonance. We showed the impact of various linker types and epitope distribution on the binding affinity to Gal-3. The synthesis of specific functionalized LacdiNAc epitopes was accomplished under the catalysis by mutant β-N-acetylhexosaminidases. The glycans were conjugated to statistic HPMA copolymer precursors through diverse linkers in a defined pattern and density using Cu(I)-catalyzed azide-alkyne cycloaddition. The resulting water-soluble and structurally flexible synthetic glyconanomaterials exhibited affinity to Gal-3 in low μM range. CONCLUSIONS: The results of this study reveal the relation between the linker structure, glycan distribution and the affinity of the glycopolymer nanomaterial to Gal-3. They pave the way to specific biomedicinal glyconanomaterials that target Gal-3 as a therapeutic goal in cancerogenesis and other disorders.

• Klíčová slova
• Carbohydrate, ELISA, Galectin-3, Glyconanomaterial, HPMA copolymer, Surface plasmon resonance,
• MeSH
• akrylamidy chemie   metabolismus   MeSH
• galektin 3 metabolismus   MeSH
• galektiny MeSH
• glykokonjugáty chemie   metabolismus   MeSH
• krevní proteiny MeSH
• lékové transportní systémy * MeSH
• lidé MeSH
• nanostruktury chemie   MeSH
• nosiče léků chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akrylamidy MeSH
• galektin 3 MeSH
• galektiny MeSH
• glykokonjugáty MeSH
• krevní proteiny MeSH
• LGALS3 protein, human MeSH Prohlížeč
• N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
• nosiče léků MeSH

Natural flavonoids, especially in their glycosylated forms, are the most abundant phenolic compounds found in plants, fruit, and vegetables. They exhibit a large variety of beneficial physiological effects, which makes them generally interesting in a broad spectrum of scientific areas. In this review, we focus on recent advances in the modifications of the glycosidic parts of various flavonoids employing glycosidases, covering both selective trimming of the sugar moieties and glycosylation of flavonoid aglycones by natural and mutant glycosidases. Glycosylation of flavonoids strongly enhances their water solubility and thus increases their bioavailability. Antioxidant and most biological activities are usually less pronounced in glycosides, but some specific bioactivities are enhanced. The presence of l-rhamnose (6-deoxy-α-l-mannopyranose) in rhamnosides, rutinosides (rutin, hesperidin) and neohesperidosides (naringin) plays an important role in properties of flavonoid glycosides, which can be considered as "pro-drugs". The natural hydrolytic activity of glycosidases is widely employed in biotechnological deglycosylation processes producing respective aglycones or partially deglycosylated flavonoids. Moreover, deglycosylation is quite commonly used in the food industry aiming at the improvement of sensoric properties of beverages such as debittering of citrus juices or enhancement of wine aromas. Therefore, natural and mutant glycosidases are excellent tools for modifications of flavonoid glycosides.

• Klíčová slova
• catechin, enzymatic hydrolysis, hesperetin, icariin, naringenin, puerarin, quercetin, rhamnosidase, rutinosidase, transglycosylation,
• MeSH
• flavonoidy metabolismus   MeSH
• glykosidhydrolasy metabolismus   MeSH
• isoflavony metabolismus   MeSH
• katechin metabolismus   MeSH
• lidé MeSH
• quercetin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• flavonoidy MeSH
• glykosidhydrolasy MeSH
• isoflavony MeSH
• katechin MeSH
• puerarin MeSH Prohlížeč
• quercetin MeSH

BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

• MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• fylogeneze MeSH
• glykosylace MeSH
• katalytická doména MeSH
• kinetika MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• reprodukovatelnost výsledků MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• Talaromyces enzymologie   MeSH
• výpočetní biologie * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH