Characterization of the putative catalytic domain of two xyloglucan xylosyltransferases (XXTs) involved in the biosynthesis of xyloglucan in Arabidopsis thaliana

Xyloglucan is the primary hemicellulosic component of the primary cell wall in dicotyledonous plants. It is characterized by a beta-(1-4)-D-glucan backbone that is substituted in a regular pattern with alpha-(1-6)-D-xylose, and can be substituted further with beta-(1-2)-D-galactose or beta-(1-2)-D-galactose-alpha(1-2)-L-fucose disaccharide. Three xyloglucan xylosyltransferases (XXTs), XXT1, XXT2, and XXT5, are responsible for the D-xylosylation of the beta-(1-4)-D-glucan backbone in Arabidopsis thaliana. They are Golgi-localized type II transmembrane proteins that have putative DXD catalytic centers. All have been shown to interact in a protein complex, XXT2 and XXT5 showing the strongest interaction, and XXT1 and XXT2 have demonstrated alpha-D-xylosyltransferase (XT) activity in vitro. The catalytic mechanism of XXT2 and XXT1, as well as the biological function of XXT5, are still unknown.;To study the functional role of DXD motifs, DXD to AXA point mutants were made in two DXD motifs (DWD and DSD) of XXT2 and XXT5. N-terminal truncated versions of these proteins were expressed in E. coli to study in vitro catalytic activity and full-length proteins were stably transformed to xxt knock-out A. thaliana to study their roles in vivo. Results demonstrate that both DWD and DSD motifs are required for XXT2 catalytic activity in vitro. Additionally, it was shown that tXXT2-His could be isolated to >90% purity using both native and hybrid conditions and exhibit XT activity.;XXT5mut(1, 2, 12):xxt5 complemented transgenic A. thaliana plants were generated, genotyped, phenotyped, analyzed for protein expression, and analyzed for XyG content in their cell walls. The results show that XXT5 with mutations in the DWD motif can complement the xxt5 phenotype while XXT5 with mutations in the DSD motif only partially complement, demonstrating that the DSD motif is essential for full XXT5 function. Though it is still unclear whether XXT5 possesses XT activity, its putative DSD catalytic center is crucial for function. It is hypothesized that either, one, XXT5 requires interaction with XXT2 (or another protein) for catalytic function or two, XXT5 does not play a direct catalytic role but promotes the activity of XXT2 or XXT1 via protein-protein interactions or by binding and stabilizing the elongating glucan backbone of XyG during xylosylation.