Enzymes that catalyze metabolic reactions in plants are highly regulated in order to prevent detrimental inefficiencies in biosynthesis and energy production. One of the most prevalent mechanisms plant cells use to regulate the activity of metabolic enzymes is enzyme-catalyzed bonding of a phosphate molecule to an amino acid within the enzyme's protein structure. This form of enzyme regulation, termed reversible protein-phosphorylation is the mechanism controlling thousands of enzymes in the cell. The plant metabolic enzyme pyruvate,phosphate dikinase (PPDK), the subject of this research, is an example of an enzyme regulated by reversible phosphorylation. PPDK catalyzes the synthesis phosphoenolpyruvate, a "building block" for a diverse set of molecules such as amino acids and cell-wall polymers. Conferring regulation of PPDK is the enzyme RP (PPDK Regulatory Protein). RP, the primary focus of this research project, had been demonstrated by previous studies to have unprecedented, one-of-a-kind catalytic properties. However, the structural basis for these properties has remained a mystery as the gene for RP had resisted cloning until this year, when the PI and coworkers isolated the gene from Arabidopsis. Their analysis of the encoded polypeptide showed that RP represents a fundamentally new kind of phosphorylating-regulatory enzyme hitherto unknown in plants or animals. This research aims to capitalize on the cloning of the RP gene by elucidating the structural basis of the enzyme's unique catalytic properties. In so doing, the poorly understood mechanism that governs RP's regulation of PPDK will also be revealed. These aims will be achieved by determining the three-dimensional structure of the Arabidopsis RP protein. Molecular and genetic tools will further establish how the two distinct RP genes represented in the Arabidopsis genome are spatially expressed in planta. Finally, a combined biochemical and molecular-genetic approach will elucidate the functional differences between these two RP enzymes. Broader impacts include (i) increasing our knowledge base in plant carbon metabolism for developing cellulosic ethanol biofuels, and (ii) the recruitment and training of undergraduate students as future scientists for our Nation's bourgeoning biotechnology sector.
