Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Olga A. Zabotina
Xyloglucan (XyG) is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants, and has important structural and physiological functions in plant growth and development. In Arabidopsis thaliana, a glucan synthase CSLC4, three xylosyltransferases, XXT1, XXT2, and XXT5, two galactosyltransferase, MUR3 and XLT2 and a fucosyltransferase, FUT1 synthesize xyloglucan in Golgi. The functional organization of these enzymes is not clear. To study the functional organization of these enzymes, Bimolecular Fluorescence Complementation (BiFC), in vitro pull-down assay and co-immunoprecipitation were used to elucidate the interactions among CSLC4, XXTs, MUR3, XLT2 and FUT1 proteins both in vivo and in vitro. Obtained results show agreement with each other and indicate the physical interactions and/or close proximity among these proteins. To further understand the stoichiometry and exact composition of the complex, the proteomics analysis of the protein complexes immunoprecipitated using different tagged glycosyltransferases as bait proteins is being performed.
The other part of this dissertation is to understand the molecular mechanism of XXTs catalytic activity. Currently, not much information is known regarding the catalytic mechanism of cell wall related glycosyltransferases due to the lack of crystal structures. We conducted homology modeling and molecular simulation to predict structure of substrate binding domain (DXD motif) of XXT2 and XXT5. Using this information, we are performing now the site-directed mutagenesis to understand the importance of XXT2 and XXT5 DXD motifs for their functionality both in vitro and in vivo.
Chou, Yi-Hsiang, "New insights into the functional organization of xyloglucan biosynthetic enzymes and their catalytic mechanism" (2014). Graduate Theses and Dissertations. 14107.