title: Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites.
abstract: BACKGROUND
  Xyloglucan 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.
  
  
  METHODS
  cDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated.
  
  
  RESULTS
  Significant differences in catalytic efficiencies (k(cat)*K(m)(-)(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.
  
  
  CONCLUSIONS
  The two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 As, Norway.
authors.forename: Gustav
authors.initials: G
authors.lastname: Vaaje-Kolstad
authors.forename: Vladimir
authors.initials: V
authors.lastname: Farkas
authors.forename: Maria
authors.initials: M
authors.lastname: Hrmova
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D012997:Solvents / Q000494:pharmacology; D014990:Xylans / Q000378:metabolism*
chemical_list: D005936:Glucans; D009844:Oligosaccharides; D012997:Solvents; D014990:Xylans; C029353:xyloglucan; D005964:Glucosyltransferases; C032411:xyloglucan 4-beta-D-glucosyltransferase