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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., Arbuscular mycorrhiza (AM) is a root endosymbiosis between fungi of the ancient phylum Glomeromycota and terrestrial plants. AM is formed by 70-90% of land plant species, improving the uptake of [...]

Endoglucanase Ce16A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Ce16A from T. fusca in complex with a nonhydrolysable structure analogue that acts as an inhibitor, methylcellobiosyl-4-thiobeta-cellobioside is determined to 1.5-angstrom resolution.

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 Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of Neu NAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria.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. [ABSTRACT FROM AUTHOR]

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis. [ABSTRACT FROM AUTHOR]

The enzymatic degradation of cellulose continues to be one of the most important enzyme-catalysed reactions. Glycoside hydrolases from family GH-6 hydrolyse cellulose with inversion of the configuration of the anomeric carbon. Whilst the catalytic proton donor has been clearly identified (Asp226 in Humicola insolens Cel6A), the identification and even the existence of a potential Brønsted base remains unclear. Equally controversial is the role of surface-loop flexibility. Here, the structure of the D416A mutant of the H. insolens cellobiohydrolase Cel6A in complex with a nonhydrolysable thiooligosaccharide methyl cellobiosyl-4-thio-β-cellobioside at 1.9 Å resolution is presented. Substrate distortion in the -1 subsite, to a ²S[sub 0] skew-boat conformation, is observed, similar to that seen in the analogous Trichoderma reesei Cel6A structure [Zou et al. (1999), Structure, 7, 1035-1045], but the active-centre N-terminal loop of the H. insolens enzyme is found in a more open conformation than described for previous structures. [ABSTRACT FROM AUTHOR]

Many three-dimensional structures of retaining β-D-glycoside hydrolases have been determined, yet oligosaccharide complexes in which the ligand spans the catalytic centre are rare. Those that have been reported so far have revealed two modes of binding: those in which the substrate adopts a distorted skew-boat or envelope conformation in the -1 subsite, reflecting the distortion observed during the catalytic cycle, and those which bypass the true catalytic centre and thus lie in a non-productive manner across the -1 subsite. The three-dimensional structure of a retaining endocellulase, Bacillus agaradhaerens Ce15A, in complex with methyl 4,4II,4III,4IV-tetrathio-α-cellopentoside falls into this latter category. The 1.1 Å structure reveals the binding of five pyranosides, all in the 4C1 chair conformation, occupying the -3, -2, +1 and +2 subsites whilst evading the catalytic machinery located in the true -1 subsite. Such binding is in marked contrast to the structure of another retaining endocellulase, the Fusarium oxysporum Ce17B, the identical ligand in which displayed a distorted skew-boat conformation at the active centre. These two binding modes may reflect different steps in the binding and catalytic process. [ABSTRACT FROM AUTHOR]

A 4 II -protected a -cellobiosyl fluoride is an efficient donor for the enzymatic stepwise condensations onto methyl b -D-glycoside acceptors. These reactions, catalyzed by the Cel7B E197A glycosynthase from Humicola insolens, gave cellodextrins from degree of polymerization 3 to 6 in high yields. [ABSTRACT FROM AUTHOR]

Investigates the implication of intramolecular fluorescence energy transfer for endo-chitinases and chitobiosidases. Assay of chitinolytic activities at namolar concentration; Determination of initial rates by time-course monitoring; Synthesis of a bifunctionalized chitooligosaccharide.

The mesophilic bacterium Clostridium cellulolyticum exports multienzyme complexes called cellulosomes to digest cellulose. One of the three major components of the cellulosome is the processive endocellulase CelF. The crystal structure of the catalytic domain of CelF in complex with two molecules of a thiooligosaccharide inhibitor was determined at 2.0 Å resolution. This is the first three-dimensional structure to be solved of a member of the family 48 glycosyl hydrolases. The structure consists of an (αα)6-helix barrel with long loops on the N-terminal side of the inner helices, which form a tunnel, and an open cleft region covering one side of the barrel. One inhibitor molecule is enclosed in the tunnel, the other exposed in the open cleft. The active centre is located in a depression at the junction of the cleft and tunnel regions. Glu55 is the proposed proton donor in the cleavage reaction, while the corresponding base is proposed to be either Glu44 or Asp230. The orientation of the reducing ends of the inhibitor molecules together with the chain translation through the tunnel in the direction of the active centre indicates that CelF cleaves processively cellobiose from the reducing to the non-reducing end of the cellulose chain. [ABSTRACT FROM AUTHOR]

Presents the structure of Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue. X-ray crystallography; Electron density map; Similarity to complex of chitobiase with its unhydrolyzed substrate.