Lucie Petrásková

β-N-acetylhexosaminidase: what's in a name…?

β-N-acetylhexosaminidases (EC 3.2.1.52, belonging to CAZy GH families 3, 20 and 84) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. GH family 20 β-N-acetylhexosaminidases, and GH family 3 and 84 β-N-acetylglucosaminidases from all kinds of organisms have been intensively studied from the point of view of their physiological roles, reaction mechanisms, structure and inhibition. Thanks to their outstanding substrate promiscuity, extracellular β-N-acetylhexosaminidases from filamentous fungi are able to cleave and transfer substrates bearing various functionalities, ranging from carboxylates, sulfates, acylations to azides, and even 4-deoxy glycosides. Thus, they have proved to be versatile biosynthetic tools for the preparation of both natural and modified hexosaminides under mild conditions with good yields.

The silymarin composition… and why does it matter???

The extract from milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin, contains a variety of flavonolignans and displays antioxidant, anti-inflammatory, immunomodulatory and hepatoprotective properties. As silybin is the main component of silymarin, the literature mainly focuses on this compound, ignoring all other components. This leads to problems in reproducibility of scientific results, as the exact composition of silymarin is often unknown and can vary to a certain degree depending on the processing, chemo-variety of the plant used and climatic conditions during the plant growth. There are studies dealing with the analytical separation and quantification of silymarin components as well as studies focused on silymarin content in clinically used drugs, in various plant parts, seasons, geographic locations etc. However, no comparison of detail flavonolignan profiles in various silymarin preparations is available to date. Also, as a result of the focus on the flavonolignans; the oil fraction, which contains linoleic, oleic and palmitic acids, sterols, tocopherol (vitamin E) and phospholipids, has been neglected. Due to all these factors, the whole plant is used e.g. as animal feed, the leaves can be eaten in salads and seed oil, besides culinary uses, can be also utilized for biodiesel or polymer production. Various HPLC separation techniques for the determination of the content of the flavonolignans have been vastly summarized in the present review.

Glycan-decorated HPMA copolymers as high-affinity lectin ligands

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.

Synthesis and Antiradical Activity of Isoquercitrin Esters with Aromatic Acids and Their Homologues

Isoquercitrin, (IQ, quercetin-3-O-β-d-glucopyranoside) is known for strong chemoprotectant activities. Acylation of flavonoid glucosides with carboxylic acids containing an aromatic ring brings entirely new properties to these compounds. Here, we describe the chemical and enzymatic synthesis of a series of IQ derivatives at the C-6″. IQ benzoate, phenylacetate, phenylpropanoate and cinnamate were prepared from respective vinyl esters using Novozym 435 (Lipase B from Candida antarctica immobilized on acrylic resin). The enzymatic procedure gave no products with “hydroxyaromatic” acids, their vinyl esters nor with their benzyl-protected forms. A chemical protection/deprotection method using Steglich reaction yielded IQ 4-hydroxybenzoate, vanillate and gallate. In case of p-coumaric, caffeic, and ferulic acid, the deprotection lead to the saturation of the double bonds at the phenylpropanoic moiety and yielded 4-hydroxy-, 3,4-dihydroxy- and 3-methoxy-4-hydroxy-phenylpropanoates. Reducing capacity of the cinnamate, gallate and 4-hydroxyphenylpropanoate towards Folin-Ciocalteau reagent was significantly lower than that of IQ, while other derivatives displayed slightly better or comparable capacity. Compared to isoquercitrin, most derivatives were less active in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, but they showed significantly better 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid, ABTS) scavenging activity and were substantially more active in the inhibition of tert-butylhydroperoxide induced lipid peroxidation of rat liver microsomes. The most active compounds were the hydroxyphenylpropanoates.

Chemoenzymatic Preparation and Biophysical Properties of Sulfated Quercetin Metabolites

Sulfated quercetin derivatives are important authentic standards for metabolic studies. Quercetin-3′-O-sulfate, quercetin-4′-O-sulfate, and quercetin-3-O-sulfate as well as quercetin-di-O-sulfate mixture (quercetin-7,3′-di-O-sulfate, quercetin-7,4′-di-O-sulfate, and quercetin-3′,4′-di-O-sulfate) were synthetized by arylsulfotransferase from Desulfitobacterium hafniense. Purified monosulfates and disulfates were fully characterized using MS and NMR and tested for their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging, Folin-Ciocalteau reduction (FCR), ferric reducing antioxidant power (FRAP), and anti-lipoperoxidant activities in rat liver microsomes damaged by tert-butylhydroperoxide. Although, as expected, the sulfated metabolites were usually less active than quercetin, they remained still effective antiradical and reducing agents. Quercetin-3′-O-sulfate was more efficient than quercetin-4′-O-sulfate in DPPH and FCR assays. In contrast, quercetin-4′-O-sulfate was the best ferric reductant and lipoperoxidation inhibitor. The capacity to scavenge ABTS+• and DMPD was comparable for all substances, except for disulfates, which were the most efficient. Quantum calculations and molecular dynamics simulations on membrane models supported rationalization of free radical scavenging and lipid peroxidation inhibition. These results clearly showed that individual metabolites of food bioactives can markedly differ in their biological activity. Therefore, a systematic and thorough investigation of all bioavailable metabolites with respect to native compounds is needed when evaluating food health benefits.

Interlaboratory Coverage Test on Plant Food Bioactive Compounds and Their Metabolites by Mass Spectrometry-Based Untargeted Metabolomics

Bioactive compounds present in plant-based foods, and their metabolites derived from gut microbiota and endogenous metabolism, represent thousands of chemical structures of potential interest for human nutrition and health. State-of-the-art analytical methodologies, including untargeted metabolomics based on high-resolution mass spectrometry, are required for the profiling of these compounds in complex matrices, including plant food materials and biofluids. The aim of this project was to compare the analytical coverage of untargeted metabolomics methods independently developed and employed in various European platforms. In total, 56 chemical standards representing the most common classes of bioactive compounds spread over a wide chemical space were selected and analyzed by the participating platforms (n = 13) using their preferred untargeted method. The results were used to define analytical criteria for a successful analysis of plant food bioactives. Furthermore, they will serve as a basis for an optimized consensus method.

Sulfated Metabolites of Flavonolignans and 2,3-Dehydroflavonolignans: Preparation and Properties

Silymarin, an extract from milk thistle (Silybum marianum) fruits, is consumed in various food supplements. The metabolism of silymarin flavonolignans in mammals is complex, the exact structure of their metabolites still remains partly unclear and standards are not commercially available. This work is focused on the preparation of sulfated metabolites of silymarin flavonolignans. Sulfated flavonolignans were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate as a sulfate donor and characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). Their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging; ferric (FRAP) and Folin–Ciocalteu reagent (FCR) reducing activity; anti-lipoperoxidant potential; and effect on the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway were examined. Pure silybin A 20-O-sulfate, silybin B 20-O-sulfate, 2,3-dehydrosilybin-20-O-sulfate, 2,3-dehydrosilybin-7,20-di-O-sulfate, silychristin-19-O-sulfate, 2,3-dehydrosilychristin-19-O-sulfate, and silydianin-19-O-sulfate were prepared and fully characterized. Sulfated 2,3-dehydroderivatives were more active in FCR and FRAP assays than the parent compounds, and remaining sulfates were less active chemoprotectants. The sulfated flavonolignans obtained can be now used as authentic standards for in vivo metabolic experiments and for further research on their biological activity.

Overproduction and characterization of the first enzyme of a new aldoxime dehydratase family in Bradyrhizobium sp.

Almost 100 genes within the genus Bradyrhizobium are known to potentially encode aldoxime dehydratases (Oxds), but none of the corresponding proteins have been characterized yet. Aldoximes are natural substances involved in plant defense and auxin synthesis, and Oxds are components of enzymatic cascades enabling bacteria to transform, utilize and detoxify them. The aim of this work was to characterize a representative of the highly conserved Oxds in Bradyrhizobium spp. which include both plant symbionts and members of the soil communities. The selected oxd gene from Bradyrhizobium sp. LTSPM299 was expressed in Escherichia coli, and the corresponding gene product (OxdBr1; GenBank: WP_044589203) was obtained as an N-His6-tagged protein (monomer, 40.7 kDa) with 30-47% identity to Oxds characterized previously. OxdBr1 was most stable at pH ca. 7.0-8.0 and at up to 30 °C. As substrates, the enzyme acted on (aryl)aliphatic aldoximes such as E/Z-phenylacetaldoxime, E/Z-2-phenylpropionaldoxime, E/Z-3-phenylpropionaldoxime, E/Z-indole-3-acetaldoxime, E/Z-propionaldoxime, E/Z-butyraldoxime, E/Z-valeraldoxime and E/Z-isovaleraldoxime. Some of the reaction products of OxdBr1 are substrates of nitrilases occurring in the same genus. Regions upstream of the oxd gene contained genes encoding a putative aliphatic nitrilase and its transcriptional activator, indicating the participation of OxdBr1 in the metabolic route from aldoximes to carboxylic acids.