Abstract:
Primary cell walls of the coniferous gymnosperm Pinus radiata are rich in xyloglucans. The principal enzymes involved in the synthesis of these polysaccharides are glycosyltransferases (GTs) which form glycosidic bonds using nucleotide sugars as donors. However, very few studies have been carried out on gymnosperm enzymes involved in cell-wall polysaccharide synthesis. This study aimed to purify, crystallize and determine the complete structure of the xyloglucan (1→6)-α-xylosyltransferase of Pinus radiata expressed heterologously. This is a glycosyltransferase family 34 protein (PrGT34B) homologous to the Arabidopsis thaliana proteins XXT1 and XXT2. No protein structures have been determined for plant glycosyltransferase family 34 proteins. N-terminal truncated PrGT34B protein was heterologously expressed in a Sf9 expression system. Significant progress was made in purifying this PrGT34B protein. Initial purification was done by immobilized metal affinity chromatography with a cobalt column and the histidine-tagged recombinant protein. Recombinant tobacco etch virus protease was effective in cleaving the histidine tag from the PrGT34B protein. Size exclusion chromatography multiangle light scattering indicated the protein formed oligomers: tetramers (~ 200 kDa) and dimers (~ 100 kDa), as well as the monomer (~ 50 kDa). The reducing agent TCEP was found to be effective in preventing the formation of most tetramers, but not dimers. Size exclusion chromatography on a S-200 column in the presence of reducing agent TCEP gave the majority of the protein in the form of dimers. Differential scanning fluorimetry (DSF) showed that the PrGT34B protein dimer unfolded at 39oC indicating the protein was unstable. However, the PrGT34B protein dimer incubated with UDP-xylose showed a temperature shift to 42oC suggesting that UDP-xylose enhances the stability of the PrGT34B protein. In vivo the behaviour of PrGT34B protein is unknown. Attempts to crystallize the apo-protein in the form of dimers were unsuccessful but crystallization may be possible in the presence of UDP-xylose.
