Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites

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.

General significance

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.     

O-oligosaccharidyl-1-amino-1-deoxyalditols as intermediates for fluorescent labelling of oligosaccharides

Reducing monosaccharides were efficiently converted to stable 1-amino-1-deoxyalditols (=glycamines; distinguished from glycosylamines by mass-spectrometry) during incubation at 20 degrees C in saturated aqueous NH(4)HCO(3) containing NaCNBH(3). Potentially useful by-products included a novel, fully-reduced dimer (the corresponding secondary glycamine) and several relatively long-lived, unreduced products. With increasing incubation time, monomers exceeded dimers. Reducing disaccharides and oligosaccharides underwent similar reactions at their reducing termini; the yield of dimers decreased with increasing oligosaccharide M(r). The O-oligosaccharidyl-1-amino-1-deoxyalditols (OADs) obtained by reductive amination of oligosaccharides reacted readily with lissamine rhodamine sulfonyl chloride to yield OAD-sulforhodamine conjugates linked by a stable sulfonamide bond. Conditions for this reaction were optimised (borate buffer, pH9.0-9.5). The highly fluorescent OAD-sulforhodamine products were purified on a C(18) cartridge. They were electrophoretically immobile at pH2.0 and 6.5, and migrated towards the anode in borate buffer, pH9.4. The OAD-sulforhodamines were amenable to TLC and were excellent substrates for enzymic transglycosylation and for glycosylhydrolase action.