Fungal rutinosidases are retaining diglycosidases that release rutinose from flavonoid glycosides, catalyzing valuable transglycosylation reactions. We systematically compare the substrate specificity and synthetic capabilities of two structurally characterized fungal rutinosidases from Aspergillus niger (AnRut) and A. oryzae (AoRut), across a panel of natural, modified, and non-natural glycosides. AnRut displays remarkable glycone promiscuity, efficiently hydrolyzing not only rutinosides and beta-glucopyranosides but also beta-galactopyranosides, beta-xylopyranosides, and alpha-L-arabinopyranosides. AnRut also processes diglycosides, such as rutinosides and isomaltosides. It catalyzes transglycosylations with non-natural glycosides as donors, glycosylating alcoholic and phenolic acceptors, including transfer of beta-D-galacto-, beta-D-xylo-, and alpha-L-arabinopyranosyl residues. In contrast, AoRut exhibits a narrower substrate range: it recognizes rutinosides and selected monoglycosides as substrates but does not hydrolyze most other diglycosides. Unexpectedly, AoRut catalyzes efficient transglycosylation using p-nitrophenyl beta-D-galactopyranoside and pNP-alpha-L-arabinopyranoside as donors, producing novel beta-galacto- and alpha-L-arabinopyranosides of various phenolic acceptors. Both enzymes also catalyze donor self-condensation products, such as pNP-beta-D-glucopyranosyl-(1 -> 3)-beta-D-glucopyranoside and pNP-beta-D-xylopyranosyl-(1 -> 3)-beta-D-xylopyranoside and pNP-beta-D-xylopyranosyl-(1 -> 2)-beta-D-xylopyranoside (only AoRut). Notably, this is the first reported case of a rutinosidase glycosylating a sugar acceptor. These findings reveal distinct glycone preferences and mechanistic features of the two enzymes and significantly expand the repertoire of glycosides accessible through fungal rutinosidases, highlighting their utility for synthesizing novel carbohydrate derivatives.

Membrane-embedded FtsH proteases play important and diverse physiological roles in prokaryotes, chloroplasts and mitochondria, but how substrates are distinguished remains unclear. The cyanobacterium Synechocystis sp. PCC 6803 contains four FtsH homologs organized into two distinct heterocomplexes and one homocomplex. The two heterocomplexes, derived from a recent gene duplication event, are the essential FtsH1/3 complex found in the cytoplasmic membrane, and the thylakoid-embedded FtsH2/3 complex, with a role in the repair of photodamaged photosystem II. Using a domain swapping approach, we demonstrate here that the transmembrane domains of FtsH1 and FtsH2 are primary determinants of the cellular location and functional differences between the FtsH1/3 and FtsH2/3 complexes, whereas the soluble AAA+ (ATPases associated with diverse cellular activities) and protease domains and the soluble linker were largely interchangeable under the conditions tested. Overall, our findings identify the transmembrane domain as an important determinant of both the location and substrate specificity of FtsH heterocomplexes and support a role for this domain in their functional evolution.

Fucosylated carbohydrates are a vital part of human nutrition. They act as prebiotics, nourishing beneficial gut bacteria and helping shape the gut microbiome. Enzymatic synthesis is a convenient method to access these molecules. Fungal α-l-fucosidases are glycoside hydrolases that naturally cleave terminal α-linked fucose residues from glycans; those with synthetic capabilities are conveniently applicable in glycoengineering. This study investigates the fucosylation potential and regioselectivity of rare GH29 α-l-fucosidases from filamentous fungi. The screening of production of putative α-l-fucosidases by fungal strains using various inducers was combined with in silico analysis. Recombinant α-l-fucosidases were produced on a large scale and characterized with respect to their substrate specificities and pH optima. A novel transfucosylating α-l-fucosidase from Aspergillus phoenicis was thus identified and characterized. It was capable of regioselective formation of an α-(1 → 2)-linked fucosylated product. These findings highlight the potential of selected fungal α-l-fucosidases as promising tools for glycoengineering of fucosylated carbohydrates.

A novel quercetin 2,3-dioxygenase from Penicillium chrysogenum, following biochemical characterization, served as the starting point for reshaping the substrate-binding cavity to alter its substrate specificity. Using a rational engineering strategy supported by computational predictive tools, we achieved high activity toward specific artificial flavonols. In all generated variants, amino acids were replaced with residues that naturally occur at the selected positions in homologous enzymes. Two variants with enlarged substrate-binding cavities exhibited improved activity toward bulkier substrates. In particular, the Y55F-F134L-M143L variant showed 20- to 1750-fold higher activity toward flavonol compounds with phenyl-based substitutions at position C-8. Conversely, one variant with a smaller substrate-binding cavity showed 15-fold higher activity toward the smaller flavonol 3,7-dihydroxyflavone. The procedure described here has implications for engineering metalloenzymes to alter their substrate specificity toward novel compounds.

Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) are glycosidases (EC 3.2.1.168) that cleave the glycosidic bond between the aglycone and the disaccharide residue rutinose. Their dual substrate specificity is reflected in their activity toward both rutin (rutinosylated) and isoquercitrin (glucosylated) substrates. The structure of rutinosidase from Aspergillus niger (AnRut) features a side tunnel that influences the enzyme hydrolytic and transglycosylation activities. We present a mutagenesis study of this side tunnel, and the compartment forming the +1 binding subsite, resulting in seven variants with different active-site entry geometries. We show that the key side tunnel residues affect the catalytic and trans-rutinosylation potential of AnRut and compare these properties with rutinosidase from Aspergillus oryzae, which has a side groove instead of a side tunnel. The trans-rutinosylation abilities of the enzymes were tested using a diverse library of acceptors. This work expands the structure-function understanding of fungal rutinosidases and underlies the hypothesis that the engineering of the side tunnel can increase the transglycosylation-to-hydrolysis ratio.

Galectins are small human proteins participating in inflammation processes, immune response, and cancerogenesis. Tandem-repeat galectins comprising Gal-4, Gal-8, and Gal-9 are a vital yet less studied part of the galectin fingerprint in cancer-related processes. The present work studies a library of prepared multivalent neo-glycoproteins decorated with poly-N-acetyllactosamine and human-milk-type oligosaccharides as ligands of this underexplored family of tandem-repeat galectins. A thorough binding evaluation by ELISA and biolayer interferometry was complemented with a detailed epitope mapping both from the galectin and the glycoconjugate viewpoints by nuclear magnetic resonance. The found interactions in the galectin binding site were correlated to in silico data from molecular modeling. The present work reveals pioneer information on the binding of tandem-repeat galectins to multivalent glycoconjugates carrying complex carbohydrate ligands and represents an invaluable starting point for the development of new high-affinity tailored ligands of tandem-repeat galectins, needed both for diagnosis and therapy.

5-Hydroxytryptamine receptor type 7 (5-HT7) receptor is a G protein-coupled receptor (GPCR) exhibiting noncanonical signaling properties. It has been shown that 5-HT7 can form stable inactive preassembled complexes with its cognate Gs protein. Structural determinants of such complex formation and the distinction between preassembled and intermediate activated complexes remain unknown. Here, we use molecular modeling and molecular dynamics simulations to determine and characterize the binding interface between this receptor and the Gs protein in both the active and preassembly complexes. Our results show key interaction patterns specific for the different states and pinpoint unique structural features distinguishing active, inactive, and preassembled states of the receptor.

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

A new class of compounds, namely highly substituted diaminocyclopentane-l-lysine adducts, have been discovered as potent inhibitors of O-GlcNAcase, an enzyme crucial for protein de-O-glycosylation. These inhibitors exhibit exceptional selectivity and reversibility and are the first example of human O-GlcNAcase inhibitors that are structurally related to the transition state of the rate-limiting step with the “aglycon” still in bond-length proximity. The ease of their preparation, remarkable biological activities, stability, and non-toxicity make them promising candidates for the development of anti-tau-phosphorylation agents holding significant potential for the treatment of Alzheimer's disease.

The synthesis of nitriles is of utmost importance for preparative organic chemistry. The classical routes are often associated with disadvantages such as toxicity of the reagents and drastic conditions. The uses of enzymes like aldoxime dehydratases (Oxds) and hydroxynitrile lyases constitute attractive benign alternatives. In this review, we summarize the recent trends regarding Oxds. Thousands of oxd genes were sequenced but less than thirty Oxds were investigated on protein level. We give an overview of these Oxds, their sequence analysis, conditions required for their overexpression, and their purification and assays. We then focus on the use of Oxds especially in multistep reactions combining the chemical or chemoenzymatic synthesis of aldoximes from different starting materials with the enzymatic dehydration of aldoximes to nitriles, possibly followed by the hydration of nitriles to amides. Progress in Oxd immobilization is also highlighted. Based on data published mainly in the last 5 years, we evaluate the industrial prospects of these enzyme processes in comparison with some other innovations in nitrile synthesis. • Aldoxime dehydratases (Oxds) are promising for cyanide-free routes to nitriles • A comprehensive overview of wet-lab explored Oxds is provided • Recent trends include combining Oxds with other enzymes or chemical catalysts

Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of β-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.

The binding of human galectins by glycomimetic inhibitors is a promising therapeutic approach. The structurally distinct group of tandem-repeat galectins has scarcely been studied so far, and there is hardly any knowledge on their ligand specificity or their inhibitory potential, particularly concerning non-natural carbohydrates. Here, we present the synthesis of a library of seven 3-O-disubstituted thiodigalactoside-derived glycomimetics and their affinity to two tandem-repeat galectins, Gal-8 and Gal-9. The straightforward synthesis of these glycomimetics involved dibutyltin oxide-catalyzed 3,3́-O-disubstitution of commercially available unprotected thiodigalactoside, and conjugation of various aryl substituents by copper-catalyzed Huisgen azide-alkyne cycloaddition (CuAAC). The inhibitory potential of the prepared glycomimetics for Gal-8 and Gal-9 was assessed, and compared with the established galectins Gal-1 and Gal-3. The introduction of C-3 substituents resulted in an over 40-fold increase in affinity compared with unmodified TDG. The structure-affinity relations within the studied series were discussed using molecular modeling. Furthermore, the prepared glycomimetics were shown to scavenge Gal-8 and Gal-9 from the surface of cancer cells. This pioneering study on the synthetic inhibitors especially of Gal-9 identified lead compounds that may be used in further biomedical research.

Free cyanide (fCN) consisting of HCN and CN- is highly hazardous. Today, removal of cyanide from industrial (mining, plating, coke-plant) wastewaters largely relies on physicochemical processes followed by microbial degradation. Enzymatic processes are gaining ground but are still at a low technological stage. The cyanide-converting enzymes of interest are primarily cyanide dihydratases (CynDs; EC 3.5.5.1.), which hydrolyze HCN to formic acid and ammonia, and cyanide hydratases (CynHs; EC 4.2.1.66), which hydrate HCN to formamide. CynHs usually have much higher specific activities and a broader pH profile especially in the alkaline region compared to CynDs. However, the product of CynH, formamide, although much less toxic than fCN, still poses a significant health risk. Therefore, it is attractive to combine the CynH with an amidase that converts formamide to formic acid and ammonia. Here we demonstrate on a laboratory scale a two-step “one-pot” detoxification of fCN (5mM) to formic acid using recombinantly produced purified enzymes − CynH from Exidia glandulosa and formamidase (EC 3.5.1.49) from Bacillus cereus. The reaction proceeds at pH 9.0-10.0, which reduces the risk of HCN escape. We also hypothesize that the cascade can be used for fCN determination after coupling an NAD-dependent formate dehydrogenase.

A new class of compounds inhibiting de-O-glycosylation of proteins has been identified. Highly substituted diaminocyclopentanes are impressively selective reversible non-transition state O-β-N-acetyl-d-glucosaminidase (O-GlcNAcase) inhibitors. The ease of preparative access and remarkable biological activities provide highly viable leads for the development of anti-tau-phosphorylation agents with a view to eventually ameliorating Alzheimer's disease.

An interdisciplinary study of microplastic (MP) contamination in the water area of Lake Onego was conducted within a project funded by the Russian Science Foundation. The aim of the project was to assess the current state of this large water body, specifically its contamination with synthetic polymer particles and the associated heavy metals, to identify the sources and areas of MP accumulation, patternsin its sedimentation and transport, and to conduct a hazard assessment of this pollution. The article provides an overview and a summary of the obtained results. Based on seasonal hydrochemical data for 2019–2021, the current state of the lake was identified and the degree of its contamination with microplastics, heavy metals, and other chemicals was determined. The highest MP content was found in the water and in the bottom sediments of the upper, most heavily contaminated, part of the Kondopoga Bay, where wastewater from the Kondopoga Pulp and Paper Mill enters the lake. Active accumulation of MP was detected in the bottom sediments of Lake Onego, its content being about twice that of the Baltic Sea. Spatial and seasonal patterns of MP distribution in the water column of Lake Onego were revealed. It has been established that with the current level of Lake Onego littoral area contamination by irregularly shaped MP particles sized about 100 microns, their negative impact on the population of invasive species, Baikal amphipod Gmelinoides fasciatus, is unlikely. Having applied SEM-EMF and Raman spectroscopy, we revealed the mechanism for mineralogical destruction of plastics driven by the crystallization and growth of microminerals, which leads to local ruptures of the plastic and accelerates its destruction. Methods for fractionation of MP particles and desorption of metals from their surface have been developed, making it possible to obtain comparable and reproducible results when analyzing the metal content on their surface. For the first time, models were suggested to simulate microplastic generation from a set of macrofragments through stochastic destruction and filtering of three-dimensional microplastic fragments on a grid. The simulation results are in good agreement with the ratios obtained during field experiments.The revealed relationships can be used to adjust the detected levels of microplastics in the nature depending on the mesh size of the net used in sampling.

Nitrilases have a high potential for application in organic chemistry, environmental technology, and analytics. However, their industrial uses require that they are produced in highly active and robust forms at a reasonable cost. Some organic syntheses catalyzed by nitrilases have already reached a high level of technological readiness. This has been enabled by the large-scale production of recombinant catalysts. Despite some promising small-scale methods being proposed, the production of cyanide-converting nitrilases (cyanide hydratase and cyanide dihydratase) is lagging in this regard. This review focuses on the prospects of cyanide(di)hydratase-based catalysts. The current knowledge of these enzymes is summarized and discussed in terms of the origin and distribution of their sequences, gene expression, structure, assays, purification, immobilization, and uses. Progresses in the production of other nitrilase catalysts are also tackled, as it may inspire the development of the preparation processes of cyanide(di)hydratases.

This paper presents the results of seasonal observations of the geochemical composition of the waters of the large tributaries of Lake Onego. The mineralogy and geochemistry of the suspended matter and the isotopic composition (oxygen-18 and deuterium) of the river waters were studied for the first time. The dependence of the chemical and isotopic compositions of the tributary water on the season and characteristics of the catchment area (swampiness and lacustrine) was revealed. It is shown that the river waters belong to the bicarbonate class of the calcium group and have low mineralization, high color and a similar composition to the main minerals of the suspended matter. It is determined that the difference between the multielement spectra of the water and suspended matter of the different rivers is closely related to the geological and geomorphological structures of river basins. It is established that the quantitative characteristics of the mineral and organic parts of the suspended matter, the ratios of the different minerals andthe size and patterning of the particles of detrital material in the tributaries differ. The change in the mineralogical and geochemical compositions of the suspended matter of each individual river over the year is insignificant. The influence of the river runoff on the formation of lake waters is manifested in the chemical composition of the lake waters. The quantitative ratios of the main ions, biogenic elements and microcomponents in lake water mainly correspond to their ratios in river waters. The mineral part of the dispersed sedimentary matter of the lake in its geochemical characteristics is close to the suspended matter of the river waters.

The aim of this work was to map the sequence space of aldoxime dehydratases (Oxds) as enzymes with great potential for nitrile synthesis. Microbes contain an abundance of putative Oxds but fewer than ten Oxds were characterized in total and only two in fungi. In this work, we prepared and characterized a new Oxd (protein gb| EEU37245.1 named OxdFv) from Fusarium vanettenii 77-13-4. OxdFv is distant from the characterized Oxds with a maximum of 36% identity. Moreover, the canonical Oxd catalytic triad RSH is replaced by R141-E187-E303 in OxdFv. R141A and E187A mutants did not show significant activities, but mutant E303A showed a comparable activity as the wild-type enzyme. According to native mass spectrometry, OxdFv contained almost 1 mol of heme per 1 mol of protein, and was composed of approximately 88% monomer (41.8 kDa) and 12% dimer. A major advantage of this enzyme is its considerable activity under aerobic conditions (25.0 +/- 4.3 U/mg for E,Z-phe-nylacetaldoxime at pH 9.0 and 55 degrees C). Addition of sodium dithionite (reducing agent) and Fe2+ was required for this activity. OxdFv favored (aryl)aliphatic aldoximes over aromatic aldoximes. Substrate docking in the ho-mology model of OxdFv showed a similar substrate specificity. We conclude that OxdFv is the first characterized Oxd of the REE type.