Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of ...Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.
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► Non hydrolytic GH-18 chitinases through mutation of catalytic aspartates. ► Chitinase mutants give high DP chitin oligomers from chitinbiose oxazoline. ► Coupling reaction is ...contaminated by uncontrolled disproportionation reaction. ► Stabilizer and its assistant not a prerequisite for oxazolinium intermediate.
Two family GH-18 chitinases mutated on the key catalytic amino acids were evaluated as “glycosynthases” for the coupling of oxazoline activated donor to chitin oligosaccharide (COs) acceptors obtained from microbial fermentation.
Bacillus circulans WL-12 chitinase A1 (
Bc ChiA1) and
Trichoderma harzanium chitinase 42 (
Th Chit42) were individually mutated on the three conserved carboxylic acids, all suggested to have a role in catalysis: the general acid/base glutamate and the two aspartates, defined as the putative stabilizer and its assistant. The mutants D200A and D202A of
Bc ChiA1, together with D170N and to a lesser extent D170A of
Th Chit42 proved to be active for chitinbiose oxazoline polymerization, and also for coupling reaction between Gal(β1
→
4) chitinbiose oxazoline and chitinpentaose at neutral pH. These mutants have additionally retained the ability to catalyze transglycosylation reaction on natural COs, whereas their hydrolytic activity is abolished. Such mutants can be considered as chitin transglycosylases, verifying also the fact that the two conserved aspartic acids, the stabilizer and its assistant, are not a prerequisite for the formation of oxazolinium intermediate by acetamido anchimeric assistance, i.e. for the substrate-assisted catalysis mechanism of family GH-18 chitinases.
Arabidopsis Xyloglucan Xylosyltransferase 5 uses an acceptor substrate similar to that of Xylosyltransferases 1 and 2 but at a lower rate.
Xyloglucan, the most abundant hemicellulosic component of ...the primary cell wall of flowering plants, is composed of a β-(1,4)-glucan backbone decorated with
d
-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (
Arabidopsis thaliana
). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher
K
m
and 9-fold lower
k
cat
compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.
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 (MD′M) and 7 (M′DM′) 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, MD′M‐“click”‐XGO 12 showed good solubility in water with interesting surfactant properties.
Surface plasmon resonance (SPR) has been used to assay the roles of amino acid residues in the substrate binding cleft of Trichoderma harzianum chitinase Chit42, which belongs to the glycoside ...hydrolase family 18 (GH-18). Nine different Chit42 variants having amino acid mutations along the binding site cleft at subsites −4 to +2 were created and characterized with regard to their affinity toward chitinous and non-chitinous oligosaccharides. The catalytically inactive Chit42 mutant E172Q was used as the template for making the additional mutations. The E172Q mutant bound chitinoligosaccharides (tetra-, penta- and hexamer) with an increasing affinity from 12 to 0.2 μM whereas no binding of chitinbiose, -triose or 3′-sialyl-N-acetyllactosamine (Neu5Acα-3Galβ-4GlcNAc) could be measured, indicative of significantly lower affinity for these shorter oligosaccharides. The strongest binding affinity was displayed toward allosamidin, a transition state analog (Kd = 3 nM), and this was shown to be dependent on the E172 residue, the acid/base catalyst of Chit42. Hydrogen bonding by the glutamic acid E317 between subsites −2 and −3 and particularly the stacking interactions by tryptophanes at subsites −3 and +2 provided to be important, as mutations to these amino acids had a substantial negative effect to the overall binding affinity. Moreover, the substrate binding specificity of Chit42 could be altered toward binding of GlcNβ-4(GlcNAc)4 by providing a counter charge through substitution of residue T133 at subsite −3 against aspartic acid. In addition, the introduction of glutamine and particularly an asparagine residue at position 133 seemed to broaden the substrate preference of Chit42 toward Galβ-4(GlcNAc)4.
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.
Chitinbiose was produced at more than 4
g
L
−1 by a high cell density culture of an
Escherichia coli strain that co-expressed the rhizobial chitinoligosaccharide synthase gene
nodC and a truncated ...form of the chitinase gene
chiA which has been designed to be functionally produced in the
E. coli cytoplasm. Chitinpentaose, which has previously been shown to be produced by the nodC protein in growing
E. coli, was formed as an intermediate that was subsequently hydrolyzed into chitinbiose by the chitinase encoded by
chiA. Chitinbiose was mainly recovered in the extracellular medium and to prevent its catabolism, the genes for the chitinbiose PTS permease had to be disrupted. When the additional gene
lgtB for
β1,4-galactosyltransferase was expressed, intracellular chitinbiose was converted into the trisaccharide Gal
β-4GlcNAc
β-4GlcNAc which could serve as acceptor for glycosyltransferase that recognize the terminal
N-acetyllactosamine structure.
Complex oligosaccharides containing α-d-xylosyl-(1→6)-β-d-glucosyl residues and unsubstituted β-(1→4)-linked d-glucosyl units were readily synthesized using enzymatic coupling catalyzed by the Cel7B ...E197A glycosynthase from Humicola insolens. Constituting this library required four key steps: (1) preparing unprotected building blocks by chemical synthesis or enzymatic degradation of xyloglucan polymers; (2) generating the donor synthon in the enzymatic coupling by temporarily introducing a lactosyl motif on the 4-OH of the terminal glucosyl units of the xylogluco-oligosaccharides; (3) synthesizing the corresponding α-fluorides, followed by their de-O-acetylation and the glycosynthase-catalyzed condensation of these donors onto various acceptors; and (4) enzymatically releasing lactose or galactose from the reaction product, affording the target molecules in good overall yields. These complex oligosaccharides proved useful for mapping the active site of a key enzyme in plant cell wall biosynthesis and modification: the xyloglucan endo-transglycosylase (XET). We also report some preliminary enzymatic results regarding the efficiency of these compounds.
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.