The role of lipo-chitooligosaccharides (LCOs) as signaling molecules that mediate the establishment of symbiotic relationships between fungi and plants is being redefined. New evidence suggests that ...the production of these molecular signals may be more of a common trait in fungi than what was previously thought. LCOs affect different aspects of growth and development in fungi. For the ectomycorrhizal forming fungi,
Laccaria bicolor
, the production and effects of LCOs have always been studied with a symbiotic plant partner; however, there is still no scientific evidence describing the effects that these molecules have on this organism. Here, we explored the physiological, molecular, and metabolomic changes in
L. bicolor
when grown in the presence of exogenous sulfated and non-sulfated LCOs, as well as the chitooligomers, chitotetraose (CO4), and chitooctaose (CO8). Physiological data from 21 days post-induction showed reduced fungal growth in response to CO and LCO treatments compared to solvent controls. The underlying molecular changes were interrogated by proteomics, which revealed substantial alterations to biological processes related to growth and development. Moreover, metabolite data showed that LCOs and COs caused a downregulation of organic acids, sugars, and fatty acids. At the same time, exposure to LCOs resulted in the overproduction of lactic acid in
L. bicolor
. Altogether, these results suggest that these signals might be fungistatic compounds and contribute to current research efforts investigating the emerging impacts of these molecules on fungal growth and development.
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 Å, 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.
Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer ...the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16–34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites –1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G = Glcβ(1→4) and X = Xylα(1→6)Glcβ(1→4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with kcat/Km values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites –2 and –3 (for backbone Glc residues) and +2′ (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal = Galβ(1→4)) is the best donor substrate with a “blocked” nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.
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=Xylalpha1,6Glcbeta1,4-)} 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.
•Chitosan oligosaccharides were clicked onto polycaprolactone backbones (PCL-g-COs).•Amphiphilic PCL-g-COs self-assembled in solution into core-shell micelles.•Core cross-clicked nanoparticles with ...–S–S– linker gave reduction sensitivity.•Glutathione-mediated DOX release of bioreducible PCL-g-COs micelles.
Chitosan-based amphiphilic graft copolymers are commonly obtained by modification of chitosan backbones with synthetic polymers hampering both bioactivity and biodegradability. In this work, we report the preparation of a series of chitosan oligosaccharide-grafted copolymers (PCL-g-COs) from coupling reactions between azide-pendent polycaprolactones (PCL-N3) and reducing-end alkynyl-modified chitosan oligosaccharides (COs-alkynyl). The resulting PCL-g-COs self-organized in water into nanoscale micelles (Rh<20nm) having a COs shell and a PCL core. Locking of the core-micelles structure employing a disulfide-containing bis-alkyne cross-linker resulted in the formation of nano-vehicles which can be degraded in response to physiological (redox) stimuli. This feature was advantageously exploited to preferentially release an anticancer drug, doxororubicin, in response to the intracellular glutathione level.
Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted ...to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16–34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic “blocked” substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Galβ(1→4), G = Glcβ(1→4), and X = Xylα(1→6)Glcβ(1→4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (ΔG0) for the formation of the covalent GalGXXXG-enzyme as 6.3–8.5 kJ/mol (1.5–2.0 kcal/mol). The data indicate that the free energy of the β(1→4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.
Chitin and peptidoglycan fragments are well recognized as pathogen associated molecular patterns (PAMPs). Long‐chain oligosaccharides of β(1→4)‐linked N‐acetyl‐D‐glucosamine (GlcNAc) units indeed ...activate plants and mammals innate immune system. However, the mechanisms underlying PAMPs perception by lysine motif (LysM) domain receptors remain largely unknown because of insufficient availability of high‐affinity molecular probes. Here, we report a two‐enzyme cascade to synthesize long‐chain β(1→4)‐linked GlcNAc oligomers. Expression of the D52S mutant of hen egg‐white lysozyme (HEWL) in Pichia pastoris at 52 mg L−1 provided a new glycosynthase catalyzing efficient polymerization of α‐chitintriosyl fluoride. Selective N‐deacetylation at the non‐reducing unit of the glycosyl fluoride donor by Sinorhizobium meliloti NodB chitin‐N‐deacetylase abolished its ability to be polymerized by the glycosynthase but not to be transferred onto an acceptor. Using NodB and D52S HEWL in a one‐pot cascade reaction allowed the synthesis on a milligram scale of chitin hexa‐, hepta‐ and octasaccharides with yields up to 65 % and a perfect control over their size.
An enzymatic cascade synthesis of long‐chain β(1→4)‐GlcNAc oligosaccharides perfectly controlled in size is reported. A hen egg‐white lysozyme glycosynthase was developed to catalyze the glycosylation of α‐chitintriosyl fluoride. Using this glycosynthase in concert with a chitin‐oligosaccharide N‐deacetylase allowed controlled glycosylation of the fluoride onto oligosaccharide acceptors without competing polycondensation reaction. This enzymatic cascade enabled the preparation of the important plant elicitors chitin hexa‐, hepta‐ and octasaccharides with very good yields.
Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune ...responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is, therefore, desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Herein, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N‐acetyl‐β‐D‐glucosaminyl‐(1→4)‐1,6‐anhydro‐N‐acetyl‐β‐D‐muramic acid (GlcNAc‐anhMurNAc), a common precursor of Gram‐negative and Gram‐positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram‐positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the in vivo production of Gram‐negative muropeptides containing meso‐diaminopimelic acid, a rarely available amino acid.
A straightforward access to Gram‐negative and Gram‐positive muropeptides was developed by a chemo‐enzymatic strategy based on metabolically engineered E.coli cells.
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-β-chitobioside. Double mutants Cel7B E197A H209A ...and Cel7B E197A H209G preferentially catalyze the formation of a β-(1→4) linkage between the two disaccharides, while single mutant Cel7B E197A and triple mutant Cel7B E197A H209A A211T produce predominantly the β-(1→3)-linked tetrasaccharide. This result constitutes the first report of the modulation of the regioselectivity through site-directed mutagenesis for an endoglycosynthase.
Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling nodulation of legumes by rhizobia. More recently LCOs were also found in ...symbiotic fungi and, more surprisingly, very widely in the kingdom fungi including in saprophytic and pathogenic fungi. The LCO-V(C18:1, Fuc/MeFuc), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants, such as Medicago truncatula, can discriminate between Nod-LCOs and these Fung-LCOs. To address this question, we performed a Genome Wide Association Study on 173 natural accessions of Medicago truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-and Fung-LCOs stimulated root branching in most accessions but the root responses to these two types of LCO molecules were not correlated. Also, heritability of root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, but only one was common. This suggests that Nod-and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.
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