1,721,067 research outputs found
Glycosynthases as tools for the production of glycan analogs of natural products
No full textOligo-, polysaccharides, and glycoconjugates are a relevant part of the bioactive components of the natural products exploited in therapeutics, diagnostics, food additives, and biomaterials. Glycans are directly involved in important biological processes, such as immunostimulation, anti-inflammatory, antioxidant, and chemoprotectant actions and/or are crucial for their activity, by modulating target recognition, stability, and pharmacokinetics. On the other hand, carbohydrate extracts used for functional studies are rather heterogeneous and lack structural information because of their intrinsic complexity hampering purification and characterization. Therefore, methods for glycoside synthesis and modification are urgently needed. Recently, glycosynthases, engineered glycoside hydrolases with no hydrolytic activity that synthesize glycans in quantitative yields, were introduced. Here we will illustrate how the glycosynthases described so far might be exploited for the production of glycan analogs of natural products and their enormous potential in this field
Translational recoding in archaea
No full textTranslational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ±1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central 'dogma' of biology that information flows as DNA→RNA→protein and that it is stored in a co-linear way between the 5'→3' of nucleic acids and N→C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed -1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life
Glycosynthases: new enzymes for oligosaccharide synthesis
No full textThe mutation of putative acidrbase and nucleophile of the active sites of retaining glycosyl hydrolases, together with kinetic analysis of the mutants, and stereochemical identification of products lead to useful information for the understanding of the reaction mechanism of these enzymes. This was the preliminary and fundamental step toward the preparation of new enzymatic activities called glycosynthases. Direct exploitation of this information has been possible, leading to the design of four new enzymes for oligosaccharides synthesis. The interest for these biocatalysts rises from the fact that the yield of the reaction can be increased and selectivity can be interpreted as key characteristic of the transfer reaction instead of a balance of hydrolytic and transferring pathways followed either by substrates and products. These new biocatalysts possess different specificities and are promising and useful tools in the construction of oligosaccharide molecules of great biological interest. This short review focused the attention on different glycosynthases obtained from four glycosyl hydrolases highlighting on the preparation and development of these new enzyme
Interrupted genes in extremophilic archaea: mechanisms of gene expression in early organisms
No full textExtremophilic Archaea populate biotopes previously considered inaccessible for life. This feature, and the possibility that they are the extant forms of life closest to the last common ancestor, make these organisms excellent candidates for the study of evolution on Earth and stimulate the exobiological research in planets previously considered totally inhospitable. Among the other aspects of the physiology of these organisms, the study of the molecular genetics of extremophilic Archaea can give hints on how the genetic information is transmitted and propagated in ancient forms of life. We review here the expression of interrupted genes in a recently discovered nanoarchaeon and the mechanisms of reprogrammed genetic decoding in Archaea
A novel, efficient and sustainable strategy for the synthesis of α-glycoconjugates by combination of a α-galactosynthase and a green solvent
No full textGlycosynthases are becoming important enzymatic tools for the synthesis of oligosaccharides. Herein, we explore for the first time the synthesis of a-glycoconjugates using a a-glycosynthase in green solvents. Using this biocatalyst, b-Gal-N3 as donor, pNP-Glc and pNP-Man as acceptors, and green co-solvents we obtained high yields and excellent selectivities in the synthesis of a-glycoconjugates. In addition, reaction scale-up is feasible and co-solvent can be recovered and reused, increasing the sustainability of the reaction process. The results demonstrate that the combination of a glycosynthase and a green solvent is a promising alternative for the synthesis of glycoconjugates. The non-hydrolytic capability of this enzyme on the product obtained is a key feature that can be expanded to other glycosynthases
Probing the catalytically essential residues of the alpha-L-fucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus
No full textRetaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist
Pharmacological chaperone therapy for lysosomal storage diseases
No full textPharmacological chaperone therapy is an emerging approach to treat lysosomal storage diseases. Small-molecule chaperones interact with mutant enzymes, favor their correct conformation and enhance their stability. This approach shows
significant advantages when compared with existing therapies, particularly in terms of the bioavailability of drugs, oral administration and positive impact on the quality of patients' lives. On the other hand, future research in this field
must confront important challenges. The identification of novel chaperones is indispensable to expanding the number of patients amenable to this treatment and to optimize therapeutic efficacy. It is important to develop new allosteric
drugs, to address the risk of inhibiting target enzymes. Future research must also be directed towards the exploitation of synergies between chaperone treatment and other therapeutic approaches
Oligosaccharide synthesis by glycosynthases
No full textOligosaccharide synthesis is becoming increasingly important to the pharmaceutical industry because these biomolecules have potential application as therapeutics. Enzymatic synthesis of oligosaccharides includes both glycosyl transferases and glycosidases. The main advantage of this approach compared with traditional chemical synthesis is the regio- and stereo-selectivity that can be achieved without the need for protecting functional groups. However, the limited availability of glycosyl transferases, the high cost of their substrates, and the poor yields of the synthetic reactions per- formed by the glycosidases hamper their use in the large-scale production of oligosaccharides. The recent advent of glycosynthases – specifically mutated glyco- sidases that efficiently synthesize oligosaccharides but do not hydrolyse them – represents a promising sol- ution to these problems. Here, we review the most recent developments in the glycosynthase approach
Enzymatic synthesis of 2-deoxy-β-glucosides and stereochemistry of β-glycosidase from Sulfolobus solfataricus on glucal
No full textThe hyperthermophilic member of family 1 of the glycosyl hydrolases, the b-glycosidase from the archaeon Sulfolobus solfataricus (Ssb-gly), has been used for an efficient synthesis of b-2-deoxyglucosides and for stereochemical studies of the reactions of glucal in the presence of alkyl and pyranosidic acceptors. Protonation of the double bond of glucal resulting in the equatorially disposed proton was observed and an indication of the protonating amino acid in the active site was obtained by the use of a mutant enzyme. The regioselectivity in the formation of b-2-deoxyglucosides of pyranosidic acceptors is different from that reported for mesophilic biocatalysts
RNA editing and modifications of RNAs might have favoured the evolution of the triplet genetic code from an ennuplet code
No full textHere we suggest that the origin of the genetic code, that is to say, the birth of first mRNAs has been triggered by means of a widespread modification of all RNAs (proto-mRNAs and proto-tRNAs), as today observed in the RNA editing and in post-transcriptional modifications of RNAs, which are considered as fossils of this evolutionary stage of the genetic code origin. We consider also that other mechanisms, such as the trans-translation and ribosome frameshifting, could have favoured the transition from an ennuplet code to a triplet code. Therefore, according to our hypothesis all these mechanisms would be reflexive of this period of the evolutionary history of the genetic code
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