Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites |
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Authors: | Gustav Vaaje-Kolstad Vladimir Farkaš Maria Hrmova Geoffrey B Fincher |
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Institution: | 1. Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway;2. Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, 84538 Bratislava, Slovakia;3. Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia |
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Abstract: | BackgroundXyloglucan xyloglucosyl transferases (EC 2.4.1.207), known as xyloglucan endotransglycosylases (XETs) use a disproportionation reaction mechanism and modulate molecular masses of xyloglucans. However, it is not known precisely how these size modulations and transfer reactions occur with polymeric acceptor substrates.MethodscDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated.ResultsSignificant differences in catalytic efficiencies (kcat·Km−1) were observed and these values were 0.01, 0.02 and 0.2 s−1·mg−1·ml for HvXET3, HvXET4 and HvXET6, respectively, using tamarind xyloglucan as a donor substrate. HPLC analyses of the reaction mixtures showed that HvXET6 followed a stochastic reaction mechanism with fluorescently or radioactively labelled tamarind xyloglucans and xyloglucan-derived oligosaccharides. The analyses from two successive reaction cycles revealed that HvXET6 could increase or decrease molecular masses of xyloglucans. In the first reaction cycle equilibrium was reached under limiting donor substrate concentrations, while xyloglucan mass modulations occurred during the second reaction cycle and depended on the molecular masses of incoming acceptors. Deglycosylation experiments indicated that occupancy of a singular N-glycosylation site was required for activity of HvXET6. Experiments with organic solvents demonstrated that HvXET6 tolerated DMSO, glycerol, methanol and 1,4-butanediol in 20% (v/v) concentrations.ConclusionsThe two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs.General significanceThe results characterise donor and acceptor binding sites in plant XET, report that HvXETs act on xyloglucan donor substrates adsorbed onto nanocrystals and that HvXETs tolerate the presence of organic solvents. |
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Keywords: | a u arbitrary units DMBA dimethylamine borane complex DMSO dimethyl sulfoxide DPM Disintegration per minute ELSD evaporative light scattering detection (detector) GH16 family 16 glycoside hydrolase IMAC Immobilized Metal Affinity Chromatography MS mass spectrometry PABA p-aminobenzoic acid PDB Protein Data Bank SE size-exclusion TXG tamarind xyloglucan XEH xyloglucan hydrolase XET xyloglucan endotransglycosylase XGOs xyloglucan-derived oligosaccharides XTH xyloglucan transglycosylase/hydrolase XXXG xyloglucan-derived heptasaccharide [Xylp(α1 6)]-Glcp(β1 4)-[Xylp(α1 6)]-Glcp(β1 4)-[Xylp(α1 6)]-Glcp(β1 4)-Glcp XXXGol reduced XXXG |
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