Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Alan M. Myers
Martha G. James
Understanding the starch biosynthetic mechanism is often hampered by the existence of many isoforms of the involved enzymes as well as numerous pleiotropic effects of mutations affecting these enzymes. This dissertation investigated interactions among starch biosynthetic enzymes, focusing on both physical association of the proteins and functional interactions in a transgenic in vivo system. Specific protein-protein interactions among the starch biosynthetic enzymes that were indicated by yeast two-hybrid experiments were confirmed directly by affinity chromatography. The functional significance of such interactions, specifically between starch synthase and starch branching enzyme, was studied using heterologous expression in the yeast Saccharomyces cerevisiae. A preliminary step in the study was to engineer yeast for production of ADP-glucose, the precursor of starch. The results clearly showed that activity of the starch synthase of SSIII/DU1 is affected differently by the identity of the particular branching enzyme isoforms with which it acts. Furthermore, the branching enzyme isoforms BEIIa and BEIIb were shown for the first time to be functionally different from each other when acting in conjunction with SSIII/DU1. In addition to the protein-protein interactions among starch biosynthetic enzymes, a novel protein was found to be interacting strongly with SSI by affinity chromatography and mass spectrometry. Identification of this protein, referred to as cell-wall invertase-like protein, may reveal a previously unknown aspect of the physiology of the starch biosynthetic system, along with a possible role of sucrose metabolism within the plastid.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Kim, Seungtaek, "Functional interactions among starch biosynthetic enzymes " (2004). Retrospective Theses and Dissertations. 960.