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In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications. [ABSTRACT FROM AUTHOR]

This study unveils an innovative method for synthesizing coumarin S-glycosides, employing original biocatalysts able to graft diverse carbohydrate structures onto 7-mercapto-4-methyl-coumarin in one-pot reactions. The fluorescence properties of the generated thio-derivatives were assessed, providing valuable insights into their potential applications in biological imaging or sensing. In addition, the synthesized compounds exhibited no cytotoxicity across various human cell lines. This research presents a promising avenue for the development of coumarin S-glycosides, paving the way for their application in diverse biomedical research areas. [ABSTRACT FROM AUTHOR]

A first access to mono‐, di‐ and tri‐(Het)arylated pyrazolo[5,1‐b]oxazoles is reported. The series were generated from a library of 3‐(Het)arylpyrazolo[5,1‐b]oxazole platforms selectively functionalized through regioselective arylation procedures. The regioselective functionalization in C‐2 and C‐7 positions was investigated. Each Palladium‐catalyzed cross‐coupling condition was optimized and a representative library of the scope and limitations of the reactions was studied. [ABSTRACT FROM AUTHOR]

Previous works have shown the existence of protein partnership, belonging to a MultiStep Phosphorelay (MSP), potentially involved in osmosensing in Populus. The first actor of this signalling pathway belongs to the histidine-aspartate kinase (HK) family, which also includes the yeast osmosensor Sln1, as well as the Arabidopsis putative osmosensor AHK1. In poplar, the homologous AHK1 protein corresponds to a pair of paralogous proteins, HK1a and HK1b, exhibiting an extracellular domain (ECD), as in Sln1 and AHK1. An ECD alignment of AHK1-like proteins, from different plant species, showed a particularly well conserved ECD and revealed the presence of a cache domain. This level of conservation suggested a functional role of this domain in osmosensing. Thus, we tested this possibility by modelling assisted mutational analysis of the cache domain of the Populus HK1 proteins. The mutants were assessed for their ability to respond to different osmotic stress and the results point to an involvement of this domain in HK1 functionality. Furthermore, since HK1b was shown to respond better to stress than HK1a, these two receptors constituted a good system to search for osmosensing determinants responsible for this difference in efficiency. With domain swapping experiments, we finally demonstrated that the cache domain, as well as the second transmembrane domain, are involved in the osmosensing efficiency of these receptors. [ABSTRACT FROM AUTHOR]

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models (1-5), [...]

Whereas the mechanism and functional aspects of PAR synthesis have been extensively characterized, the present understanding of the PAR-degradation pathways catalysed by PARG is comparatively poor. Until now, the catalytic [...], Post-translational modification of proteins by poly(ADP-ribosyl) ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis (1). Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesized from NAD by PAR polymerases (1,2). PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death (3,4). Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family (5,6). High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl) ation and potentially of how the defects in this regulation are linked to human disease.

Author(s): Mary Ortmayer [1]; Pierre Lafite [1]; Binuraj R. K. Menon [1]; Tewes Tralau [1]; Karl Fisher [1]; Lukas Denkhaus [1]; Nigel S. Scrutton [1]; Stephen E. J. Rigby [1]; [...]

Arbutin, a natural glycoside, is well known as a commercial tyrosinase inhibitor, and thus, to prevent pigmentary disorders of skin. In fact, tyrosinase is involved in the biosynthesis of melanin, the skin main pigment. However, arbutin is subject to hydrolysis, which limits its bioactivity. In general, thioglycosides are known to be very resistant to both chemical and enzymatic hydrolysis, which increases the interaction time with their biological targets. A biocatalytic approach allowed us to access to thioglycosidic analogs of arbutin in a green approach with good to excellent yields. Such compounds have then been tested as tyrosinase inhibitors as well as inhibitors of melanin transfer from melanocytes to keratinocytes. This latter mechanism takes place via lectin (or lectin-like) receptors present on the cells surface. p-Aminophenyl β-D-thiogalactopyranoside appears to be an excellent candidate thanks to its tyrosinase inhibitory activity comparable to arbutin, while having the ability to interact with glycan receptors allowing to reduce melanin transfer. [ABSTRACT FROM AUTHOR]

The synthesis of fused bicyclic oxazolidin‐2‐one (OZO) iminosugars was achieved through a tandem Staudinger reduction/retro‐Michael/intramolecular Michael addition from the corresponding OZO azidosugars. Those precursors, based on both aldopentofuranoses and ketofuranohexoses backbones, were obtained following alkylation and oxidation of the related oxazolidine‐2‐thione (OZT) azidosugars. Eight OZO based iminosugars were isolated in good yields and evaluated for their potency as glycosidases inhibitors, thus extending the family of known 1‐Deoxynojirimycin (DNJ) bicyclic analogues. [ABSTRACT FROM AUTHOR]

The oxidation of six derivatives of terfenadone by recombinant human CYP2J2 (CYP = cytochrome P450) is examined. Results reveal that CYP2J2 showed an unusual regioselectivity in favor of the hydroxylation of the substrate terminal chain at the weakly reactive homobenzylic position.

D-Glycopyranosyl glycerols are common natural products and exhibit strong biological properties, notably as moisturizing agents in cosmetics. Their chemical synthesis remains tedious thus decreasing their potential industrial and economic development, as well as the study of their structure-function relationships. In this work, the chemo-enzymatic synthesis of three enantiopure 3-O-(β-D-glycopyranosyl)-sn-glycerols was efficiently performed using an original glycosidase from Dictyoglomus thermophilum and their preservatives properties were assessed using a challenge test method. Amongst them, the 3-O-(β-D-glucopyranosyl)-sn-glycerol exhibited a specific anti-fungus activity. [ABSTRACT FROM AUTHOR]

Terminal 'high-mannose oligosaccharides' are involved in a broad range of biological and pathological processes, from sperm-egg fusion to influenza and human immunodeficiency virus infections. In spite of many efforts, their synthesis continues to be very challenging and actually represents a major bottleneck in the field. Whereas multivalent presentation of mannopyranosyl motifs onto a variety of scaffolds has proven to be a successful way to interfere in recognition processes involving high-mannose oligosaccharides, such constructs fail at reproducing the subtle differences in affinity towards the variety of protein receptors (lectins) and antibodies susceptible to binding to the natural ligands. Here we report a family of functional high-mannose oligosaccharide mimics that reproduce not only the terminal mannopyranosyl display, but also the core structure and the branching pattern, by replacing some inner mannopyranosyl units with triazole rings. Such molecular design can be implemented by exploiting 'click' ligation strategies, resulting in a substantial reduction of synthetic cost. The binding affinities of the new 'click' high-mannose oligosaccharide mimics towards two mannose specific lectins, namely the plant lectin concanavalin A (ConA) and the human macrophage mannose receptor (rhMMR), have been studied by enzyme-linked lectin assays and found to follow identical trends to those observed for the natural oligosaccharide counterparts. Calorimetric determinations against ConA, and X-ray structural data support the conclusion that these compounds are not just another family of multivalent mannosides, but real 'structural mimics' of the high-mannose oligosaccharides. [ABSTRACT FROM AUTHOR]

Glycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining α-L-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak α-L-fucosidase and β-D-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-D-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate α-L-fucose from binding efficiently to the fucosidase- like active site relative to other GH29 enzymes. β-D-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewisx antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6-7 A apart. [ABSTRACT FROM AUTHOR]

CE was used for the first time to study the two-substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine-5′-triphosphate were in-capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine-5′-triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero-potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine-5′-diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose-1,6-bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in-capillary mixed using an electrophoretically mediated microanalysis approach. [ABSTRACT FROM AUTHOR]

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.

Galactofuranose is a rare form of the well-known galactose sugar, and its occurrence in numerous pathogenic micro-organisms makes the enzymes responsible for its biosynthesis interesting targets. Herein, we review the role of these carbohydrate-related proteins with a special emphasis on the galactofuranosidases we recently characterized as an efficient recombinant biocatalyst. [ABSTRACT FROM AUTHOR]

Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or thioglycoligation. In both cases, the release of the thioimidoyl aglycone was remotely activated through a protonation driven by a carboxylic residue in the active site of the corresponding enzymes. A recombinant glucosidase (DtGly) from Dictyoglomus themophilum, previously used in biocatalysis, was also able to use such glycosyl thioimidates as substrates. Yet, enzymatic kinetic values analysis, coupled to mutagenesis and in silico modelling of DtGly/substrate complexes demonstrated that the release of the thioimidoyl moiety during catalysis is only driven by its leaving group ability, without the activation of a remote protonation. In the search of efficient glycosyl donors, glycosyl thioimidates are attractive and efficient. Their utility, however, is limited to enzymes able to promote leaving group release by remote activation. [ABSTRACT FROM AUTHOR]