No abstract

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.

The n‐3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exert hypolipidemic effects and prevent development of obesity and insulin resistance in animals fed high‐fat diets. We sought to determine the efficacy of α‐substituted DHA derivatives as lipid‐lowering, antiobesity, and antidiabetic agents. C57BL/6 mice were given a corn oil‐based high‐fat (35% weight/weight) diet (cHF), or cHF with 1.5% of lipids replaced with α‐methyl DHA ethyl ester (Substance 1), α‐ethyl DHA ethyl ester (Substance 2), α,α‐di‐methyl DHA ethyl ester (Substance 3), or α‐thioethyl DHA ethyl ester (Substance 4) for 4 months. Plasma markers of glucose and lipid metabolism, glucose tolerance, morphology, tissue lipid content, and gene regulation were characterized. The cHF induced obesity, hyperlipidemia, impairment of glucose homeostasis, and adipose tissue inflammation. Except for Substance 3, all other substances prevented weight gain and Substance 2 exerted the strongest effect (63% of cHF‐controls). Glucose intolerance was significantly prevented (∼67% of cHF) by both Substance 1 and Substance 2. Moreover, Substance 2 lowered fasting glycemia, plasma insulin, triacylglycerols, and nonesterified fatty acids (73, 9, 47, and 81% of cHF‐controls, respectively). Substance 2 reduced accumulation of lipids in liver and skeletal muscle, as well as adipose tissue inflammation associated with obesity. Substance 2 also induced weight loss in dietary obese mice. In contrast to DHA administered either alone or as a component of the EPA/DHA concentrate (replacing 15% of dietary lipids), Substance 2 also reversed established glucose intolerance in obese mice. Thus, Substance 2 represents a novel compound with a promising potential in the treatment of obesity and associated metabolic disturbances.

Novel conjugates of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers tethered with chitooligosaccharidic epitopes of varying lengths were shown to be potent ligands of a model lectin, wheat germ agglutinin (WGA). The azide-functionalized oligosaccharidic epitopes were prepared by the action of Tyr470Asn mutant β-N-acetylhexosaminidase from Talaromyces flavus in a single reaction step and were conjugated to HPMA copolymer precursors in a defined pattern and density through Cu + -catalyzed azide-alkyne cycloaddition. The soluble, biocompatible, and structurally flexible synthetic glycopolymers were studied for their binding to WGA in a competitive enzyme-linked lectin assay (ELLA), and the kinetics of interaction were analyzed by surface plasmon resonance (SPR). To the best of our knowledge, this study presents the first HPMA copolymers derivatized with long oligosaccharides that demonstrate high affinity to a lectin target. The binding affinities in the low nanomolar and subnanomolar ranges place the prepared glycopolymers among the best WGA ligands reported to date. This study demonstrates the targeting potential of these glycopolymers for therapeutically relevant lectins. † Electronic supplementary information (ESI) available: Structural characterization of functionalized chitooligomers 3, 5-9 (NMR data and spectra, MS spectra, HPLC chromatograms); structural identification of chitooligomer standards 26-29; synthesis of polymer precursors 12a and 12b; structural characterization of glycopolymers 13-25 (NMR spectra). See a The molecular weights (M n ) and dispersities (Đ) of the polymers were determined using GPC with MALS and RI detection. b Average number of glycans per polymer chain (glycan content, mol%); n = 1, monovalent standard. c Relative potency, i.e. IC 50 (monovalent standard)/IC 50 (multivalent glycopolymer). d Relative potency per glycan.

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.

Thes ynthesis of oligosaccharidesu sing mutant glycosidases hasb een dynamically developing due to the need for novelc arbohydrate-based materials.C hitooligomers (b-1!4-linkedo ligomers of N-acetylglucosamine) are bioactive compounds applicablei nm any industrial andp harmacological areas;h owever, their accessibility is still ratherl ow. In this work, GH20 b-N-acetylhexosaminidase from the fungus Talaromyces flavus wase ngineered by site-directed mutagenesis to obtain three efficiently transglycosylating variants with ca. 200-timess uppressedh ydrolytic activity.T hus,w eh ave prepared the first GH20 transglycosidases.Inthe reactions cat-alyzed by these mutant b-N-acetylhexosaminidases we were able to easily prepare andi solate bothn atural and modified chitooligomers in sufficient amountsf or their complete spectral characterization and possible further application. Thep resented method for the synthesis of chitooligomers with aglycones suitablef or linkingt oo ther biological structures is simple and robust enough to be easily scaled up.

beta-N-Acetylhexosaminidases feature so-called wobbling specificity, which means that they cleave substrates both in gluco- and galacto- configurations, with the activity ratio depending on the enzyme source. Here we present the new finding that fungal beta-N-acetylhexosaminidases are able to hydrolyze and transfer 4-deoxy-N-acetylhexosaminides with high yields. This clearly demonstrates that the 4-hydroxy moiety at the substrate pyranose ring is not essential for substrate binding to the enzyme active site, which was also confirmed by molecular docking of the tested compounds into the model of the active site of beta-N-acetylhexosaminidase from Aspergillus oryzae. A set of four 4-deoxy-N-acetylhexosaminides was synthesized and screened against a panel of beta-N-acetylhexosaminidases (extracellular and intracellular) from various sources (fungal, human, animal, plant and bacterial) for hydrolysis. The results of this screening are reported here, as well as the structures of three novel 4'-deoxy-disaccharides prepared by transglycosylation reaction with high yields (52% total disaccharide fraction) using beta-N-acetylhexosaminidase from Talaromyces flavus.

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.