The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza.

Arbuscular mycorrhiza (AM) is a root endosymbiosis between plants and glomeromycete fungi. It is the most widespread terrestrial plant symbiosis, improving plant uptake of water and mineral nutrients. Yet, despite its crucial role in land ecosystems, molecular mechanisms leading to its formation are just beginning to be unravelled. Recent evidence suggests that AM fungi produce diffusible symbiotic signals. Here we show that Glomus intraradices secretes symbiotic signals that are a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs), which stimulate formation of AM in plant species of diverse families (Fabaceae, Asteraceae and Umbelliferae). In the legume Medicago truncatula these signals stimulate root growth and branching by the symbiotic DMI signalling pathway. These findings provide a better understanding of the evolution of signalling mechanisms involved in plant root endosymbioses and will greatly facilitate their molecular dissection. They also open the way to using these natural and very active molecules in agriculture.

Chemoenzymatic Syntheses of Sialylated Oligosaccharides Containing C5-Modified Neuraminic Acids for Dual Inhibition of Hemagglutinins and Neuraminidases.

A fast chemoenzymatic synthesis of sialylated oligosaccharides containing C5-modified neuraminic acids is reported. Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of NeuNAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria. GM3 analogues were either obtained by chemoselective modification of biosynthetic N-acetyl-sialyllactoside (GM3 NAc) or by direct bacterial synthesis using C5-modified neuraminic acid precursors. The latter strategy proved to be very versatile as it led to an efficient synthesis of GM2 analogues. These glycomimetics were assessed against hemagglutinins and sialidases. In particular, the GM3 NPhAc displayed a binding affinity for Maackia amurensis agglutinin (MAA) similar to that of GM3 NAc, while being resistant to hydrolysis by Vibrio cholerae (VC) neuraminidase. A preliminary study with influenza viruses also confirmed a selective inhibition of N1 neuraminidase by GM3 NPhAc, suggesting potential developments for the detection of flu viruses and for fighting them.

CGTase-catalysed cis-glucosylation of L-rhamnosides for the preparation of Shigella flexneri 2a and 3a haptens.

We report the enzymatic synthesis of α-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside and α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside by using a wild-type transglucosidase in combination with glucoamylase and glucose oxidase. It was shown that Bacillus circulans 251 cyclodextrin glucanotransferase (CGTase, EC 2.1.4.19) can efficiently couple an α-L-rhamnosyl acceptor to a maltodextrin molecule with an α-(1→4) linkage, albeit in mixture with the α-(1→3) regioisomer, thus giving two glucosylated acceptors in a single reaction. Optimisation of the CGTase coupling reaction with ß-cyclodextrin as the donor substrate and methyl or allyl α-L-rhamnopyranoside as acceptors resulted in good conversion yields (42-70%) with adjustable glycosylation regioselectivity. Moreover, the efficient chemical conversion of the products of CGTase-mediated cis-glucosylation into protected building blocks (previously used in the synthesis of O-antigen fragments of several Shigella flexneri serotypes) was substantiated. These novel chemoenzymatic strategies towards useful, convenient intermediates in the synthesis of S. flexneri serotypes 2a and 3a oligosaccharides might find applications in developments towards synthetic carbohydrate-based vaccine candidates against bacillary dysentery.

Host-specific Nod-factors associated with Medicago truncatula nodule infection differentially induce calcium influx and calcium spiking in root hairs.

Rhizobial nodulation (Nod) factors activate both nodule morphogenesis and infection thread development during legume nodulation. Nod factors induce two different calcium responses: intra-nuclear calcium oscillations and a calcium influx at the root hair tip. Calcium oscillations activate nodule development; we wanted to test if the calcium influx is associated with infection. Sinorhizobium meliloti nodL and nodF mutations additively reduce infection of Medicago truncatula. Nod-factors made by the nodL mutant lack an acetyl group; mutation of nodF causes the nitrogen (N)-linked C16:2 acyl chain to be replaced by C18:1. We tested whether these Nod-factors differentially induced calcium influx and calcium spiking. The absence of the NodL-determined acetyl group greatly reduced the induction of calcium influx without affecting calcium spiking. The calcium influx was even further reduced if the N-linked C16:2 acyl group was replaced by C18:1. These additive effects on calcium influx correlate with the additive effects of mutations in nodF and nodL on legume infection. Infection thread development is inhibited by ethylene, which also inhibited Nod-factor-induced calcium influx. We conclude that Nod-factor perception differentially activates the two developmental pathways required for nodulation and that activation of the pathway involving the calcium influx is important for efficient infection.

Synthesis of sulfonamide-bridged glycomimetics.

A flexible and short synthesis of sulfonamide-bridged di-, tri-, tetra-, and octasaccharide glycomimetics was accomplished by reaction of glycosyl thioacetates with amino sugar substrates. The chemistry to incorporate the sulfonamide linker in place of a native O-glycosidic bond was broadly scoped, allowing access to head-to-head (1↔1) and head-to-tail (1→2), (1→3), (1→4), and (1→6) sulfonamide-bridged glycomimetics. The synthesis proceeds with retention of configuration at the anomeric center and is compatible with variable stereochemical arrangements and with acid- and base-labile protecting groups.

Enthalpic studies of xyloglucan-cellulose interactions.

We report a study of xyloglucan (XG)-cellulose interactions made possible by the preparation of various well-defined cellulosic and xyloglucosidic substrates. Bacterial microcrystalline cellulose (BMCC) as well as cellulose whiskers (CellWhisk) were used as cellulosic substrates. Xyloglucosidic substrates were obtained from Rubus cells and Tamarindus indica seeds. Different primary structure characteristics of XGs such as the backbone length and the nature of the side chains, as well as their repartition, were considered in order to examine the influence of the primary structure on their interaction capacity. Two complementary approaches were carried out: first, the determination of adsorption isotherms and its associated models, and second, an enthalpic study using isothermal titration calorimetry (ITC). This study highlighted that an increase of XG interaction capacity occurred with increasing XG molecular weight. Furthermore, we determined that a minimum of 12 glucosyl residues on the backbone is required to observe significant interactions. Moreover, both the presence of trisaccharidic side chains with fucosyl residues and an increase of unsubstituted glucosyl residues enhanced XG-cellulose interactions. The evolution of adsorption isotherms with temperature and ITC measurements showed that two different processes were occurring, one exothermic and one endothermic, respectively. Although the presence of an exothermic interaction mechanism has long been established, the presence of an endothermic interaction mechanism has never been reported.

Microbial oligosaccharides differentially induce volatiles and signalling components in Medicago truncatula.

Plants perceive biotic stimuli by recognising a multitude of different signalling compounds originating from the interacting organisms. Some of these substances represent pathogen-associated molecular patterns, which act as general elicitors of defence reactions. But also beneficial microorganisms like rhizobia take advantage of compounds structurally related to certain elicitors, i.e. Nod-factors, to communicate their presence to the host plant. In a bioassay-based study we aimed to determine to what extent distinct oligosaccharidic signals are able to elicit overlapping responses, including the emission of volatile organic compounds which is mainly considered a typical mode of inducible indirect defence against herbivores. The model legume Medicago truncatula Gaertn. was challenged with pathogen elicitors (beta-(1,3)-beta-(1,6)-glucans and N,N',N'',N'''-tetraacetylchitotetraose) and two Nod-factors, with one of them being able to induce a nodulation response in M. truncatula. Single oligosaccharidic elicitors caused the emission of volatile organic compounds, mainly sesquiterpenoids. The volatile blends detected were quite characteristic for the applied compounds, which could be pinpointed by multivariate statistical methods. As potential mediators of this response, the levels of jasmonic acid and salicylic acid were determined. Strikingly, neither of these phytohormones exhibited changing levels correlating with enhanced volatile emission. All stimuli tested caused an overproduction of reactive oxygen species, whereas nitric oxide accumulation was only effected by elicitors that were equally able to induce volatile emission. Thus, all signalling compounds tested elicited distinct reaction patterns. However, similarities between defence reactions induced by herbivory and pathogen-derived elicitors could be ascertained; but also Nod-factors were able to trigger defence-related reactions.

Chromogenic substrates for feruloyl esterases.

Chromogenic mono- and diferuloyl-butanetriol analogs were prepared by chemical syntheses and their efficiency was evaluated as substrates for feruloyl esterases from Aspergillus niger.

Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase mutants.

Four Humicola insolens Cel7B glycoside hydrolase mutants have been evaluated for the coupling of lactosyl fluoride on O-allyl N(I)-acetyl-2(II)-azido-beta-chitobioside. Double mutants Cel7B E197A H209A and Cel7B E197A H209G preferentially catalyze the formation of a beta-(1-->4) linkage between the two disaccharides, while single mutant Cel7B E197A and triple mutant Cel7B E197A H209A A211T produce predominantly the beta-(1-->3)-linked tetrasaccharide. This result constitutes the first report of the modulation of the regioselectivity through site-directed mutagenesis for an endoglycosynthase.

Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase.

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=[Xylalpha1,6]Glcbeta1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.

A highly acid-stable and thermostable endo-beta-glucanase from the thermoacidophilic archaeon Sulfolobus solfataricus.

The thermoacidophilic archaeon Sulfolobus solfataricus P2 encodes three hypothetic endo-beta-glucanases, SSO1354, SSO1949 and SSO2534. We cloned and expressed the gene sso1949 encoding the 334 amino acids containing protein SSO1949, which can be classified as a member of glycoside hydrolase family 12. The purified recombinant enzyme hydrolyses carboxymethylcellulose as well as cello-oligomers, with cellobiose and cellotriose as main reaction products. By following the hydrolysis of a fluorescently labelled cellohexaoside under a wide variety of conditions, we show that SSO1949 is a unique extremophilic enzyme. This archaeal enzyme has a pH optimum of approx. pH 1.8 and a temperature optimum of approx. 80 degrees C. Furthermore, the enzyme is thermostable, with a half-life of approx. 8 h at 80 degrees C and pH 1.8. The thermostability is strongly pH-dependent. At neutral pH, the thermal inactivation rate is nearly two orders of magnitude higher than at pH 1.8. Homology modelling suggests that the catalytic domain of SSO1949 has a similar fold to other mesophilic, acidophilic and neutral cellulases. The presence of a signal peptide indicates that SSO1949 is a secreted protein, which enables S. solfataricus to use cellulose as an external carbon source. It appears that SSO1949 is perfectly adapted to the extreme environment in solfataric pools. A cellulolytic enzyme with such a combination of stability and activity at high temperatures and low pH has not been described so far and could be a valuable tool for the large-scale hydrolysis of cellulose under acidic conditions.

The structure of barley alpha-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: a pair of sugar tongs.

Though the three-dimensional structures of barley alpha-amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other alpha-amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in alpha-amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.

Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens.

The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7-1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases.

The beta-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 beta-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.

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D.

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

Electrochemical detection of Arachis hypogaea (peanut) agglutinin binding to monovalent and clustered lactosyl motifs immobilized on a polypyrrole film.

Direct detection of peanut agglutinin/lactose interactions was realized by an electrochemical approach based on a polypyrrole coated electrode displaying pendant carbohydrates.

Biosynthesis of conjugatable saccharidic moieties of GM2 and GM3 gangliosides by engineered E. coli.

Oligosaccharidic moieties of GM(2) and GM(3) gangliosides bearing an allyl or a propargyl aglycon, are efficiently biosynthesized on the gram scale by growing metabolically engineered Escherichia coli cells in the presence of the corresponding lactoside acceptors and sialic acid.

Chemoenzymatic synthesis of 6omega -modified maltooligosaccharides from cyclodextrin derivatives.

Regioselectively substituted maltooligosaccharides were prepared by enzymatic transformation of modified cyclodextrins by using simultaneously two different enzymes: cyclodextrin glucanotransferase (CGTase) and amyloglucosidase. Oligosaccharides were obtained in very good yields and their structures were identified by 1D and 2D NMR spectroscopy. These results provided new information about the specificity of the catalytic sites of CGTase and amyloglucosidase. They also offered new ways for the synthesis of regioselectively modified maltooligosaccharides.

High-affinity nod factor binding site from Phaseolus vulgaris cell suspension cultures.

The lipo-chitooligosaccharidic Nod factors produced by rhizobia are key molecules in the establishment of symbiosis with legumes and probably are recognized by the host plant via specific receptors. Here, we report on the presence of a binding site in cell cultures of Phaseolus vulgaris displaying a high affinity for Nod factors from Rhizobium tropici (NodRt-V) (Me, S, C18:1), a symbiont of this legume. The binding site shares common properties with NFBS2, a Nod-factor binding site previously characterised in Medicago varia, in terms of affinity, preferential plasma-membrane location, and sensitivity to proteases and lysine reactive reagents. However, the bean site poorly recognizes the Nod factors produced by Sinorhizobium meliloti, the symbiont of Medicago. The study of selectivity toward the Nod factors reveals that the length and degree of unsaturation of the acyl chain and the length of the oligosaccharidic moiety are important determinants of high affinity binding to the bean site; whereas, the N-methyl and O-sulfuryl groups play a minor role. Thus, the common characteristics of P. vulgaris and M. varia Nod-factor binding sites suggest that they probably correspond to structurally related proteins, but their different selectivity suggests that they may be involved in a differential perception system for Nod factors in legumes.