This report describes an efficient chemoenzymatic synthesis of a variety of regioselectively modified β(1→4)-oligo- and -polysaccharides. This successful approach was based on: (i) the use of a ...“glycosynthase” which is a Glu-197-Ala nucleophile mutant of the retaining cellulase endoglucanase I (Cel7B) from Humicola insolens and (ii) the rational design of modified acceptor and donor molecules through a careful examination of information given by the X-ray structures of wild type and mutated enzymes. The mutant was able to catalyze, in high yield, the regio- and stereoselective glycosylation of α-glycobiosyl fluorides both unsubstituted and modified with various mono- and disaccharide acceptors, as well as the polymerization of these donors through a single-step inverting mechanism.
Background: Myrosinase is the enzyme responsible for the hydrolysis of a variety of plant anionic 1-thio-
β-
D-glucosides called glucosinolates. Myrosinase and glucosinolates, which are stored in ...different tissues of the plant, are mixed during mastication generating toxic by-products that are believed to play a role in the plant defence system. Whilst
O-glycosidases are extremely widespread in nature, myrosinase is the only known
S-glycosidase. This intriguing enzyme, which shows sequence similarities with
O-glycosidases, offers the opportunity to analyze the similarities and differences between enzymes hydrolyzing
S- and
O-glycosidic bonds.
Results: The structures of native myrosinase from white mustard seed (
Sinapis alba) and of a stable glycosyl–enzyme intermediate have been solved at 1.6 Å resolution. The protein folds into a (
β/
α)
8-barrel structure, very similar to that of the cyanogenic
β-glucosidase from white clover. The enzyme forms a dimer stabilized by a Zn
2+ ion and is heavily glycosylated. At one glycosylation site the complete structure of a plant-specific heptasaccharide is observed. The myrosinase structure reveals a hydrophobic pocket, ideally situated for the binding of the hydrophobic sidechain of glucosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replacement of the general acid/base glutamate by a glutamine residue, the catalytic machinery of myrosinase is identical to that of the cyanogenic
β-glucosidase. The structure of the glycosyl–enzyme intermediate shows that the sugar ring is bound via an
α-glycosidic linkage to Glu409, the catalytic nucleophile of myrosinase.
Conclusions: The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst. The replacement of the general acid/base glutamate of
O-glycosidases by a glutamine residue in myrosinase suggests that for hydrolysis of the glycosyl–enzyme, the role of this residue is to ensure a precise positioning of a water molecule rather than to provide general base assistance.
Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate ...pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2.
Heterologous expression of two fungal chitinases, Chit33 and Chit42, from
Trichoderma harzianum was tested in the different compartments and on the surface of
Escherichia coli cells. Our goal was to ...find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on
E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.
The β-glucan-binding protein (GBP) of soybean (
Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a ...microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze β-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.
3II-O-Allyl-α-laminaribiosyl fluoride was prepared as a key synthon for the enzymatic synthesis of β(1rightwards arrow3)-glucan oligosaccharides, catalyzed by a mutated β(1rightwards ...arrow3)-glucanase (E231G) from barley (Hordeum vulgare L.). A strategy was developed for enzymatic elongation of the β(1rightwards arrow3)-glucan chain from the reducing end, using a single glucoside acceptor. When β-glucoside phenyl disulfide was used as the acceptor, this methodology generated laminari-oligosaccharides conjugatable at both their reducing and non-reducing ends.
Myrosinase, a thioglucoside glucohydrolase, is the only enzyme able to hydrolyse glucosinolates, a unique family of molecules bearing an anomeric O-sulfated thiohydroximate function. Non-hydrolysable ...myrosinase inhibitors have been devised and studied for their biological interaction. Diverse modifications of the O-sulfate moiety did not result in a significant inhibitory effect, whereas replacing the D-glucopyrano residue by its carba-analogue allowed inhibition to take place. X-Ray experiments carried out after soaking allowed for the first time inclusion of a non-hydrolysable inhibitor inside the enzymatic pocket. Structural tuning of the aglycon part in its pocket is being used as a guide for the development of simplified and more potent inhibitors.
Crystals of the inactive mutant Glu257→Ala of cyclodextrin glycosyltransferase were soaked with the cyclodextrin (CD) derivative S-(α-d-glucopyranosyl)-6-thio-β-CD. The structural analysis showed its ...β-CD moiety with no density indication for the exocyclic glucosyl unit. For steric reasons, however, the position of this unit is restricted to be at only two of the seven glucosyl groups of β-CD. The analysis indicated that the enzyme can cyclize branched α-glucans. The ligated β-CD moiety revealed how the enzyme binds its predominant cyclic product. The conformation of the ligated β-CD was intermediate between the more symmetrical conformation in β-CD dodecahydrate crystals and the conformation of a bound linear α-glucan chain. Its scissile bond was displaced by 2.8 Å from the position in linear α-glucans. Accordingly, the complex represents the situation after the cyclization reaction but before diffusion into the solvent, where a more symmetrical conformation is assumed, or the equivalent state in the reverse reaction. Furthermore, a unifying nomenclature for oligosaccharide-binding subsites in proteins is proposed.
Well-defined glyco-polyorganosiloxanes were synthesized by the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction (often simply referred to as click chemistry). N-propargylglycosylamines 2 ...and 4 were first synthesized from cellobiose (1) and xylogluco-oligosaccharide XGOs 3 without protecting groups. The azide function was introduced into polydimethylsiloxanes PDMS: 5 (MDM) and 7 (MDM) by azidolysis of the counterpart epoxy silicon with NaN3 to afford the mono-azido 6 and di-azido 8 derivatives, respectively. The coupling reaction took place in a hydro-alcoholic medium in the presence of CuSO4/sodium ascorbate as catalyst. Only one compound, MDM-click-XGO 12 showed good solubility in water with interesting surfactant properties.
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