Molecular characterisation of an ADP-dependent regulatory protein

Conversion of carbon dioxide to sugars by plants is essential to life on earth. In the majority of plants this process competes with non-productive reaction with oxygen. One group of plants, the C4 plants, have developed a strategy to avoid this: this strategy allows the plants to survive in hot, dry and arid conditions which would not otherwise be viable for plant growth. These plants include the common crop plants, maize and sugarcane. Research is presently underway to determine if this pathway can be engineered into other plants to generate hardier crop plants and this research will feed into this objective. Our research addresses how one part of this alternative strategy of making sugars is controlled. In particular, we are interested in how an unusual protein acts to switch off the pathway in the dark. The protein, pyruvate,orthophosphate dikinase (PPDK) regulatory protein, or PDRP for short, inactivates another protein, PPDK, thereby preventing the plant from making the compound phosphoenolpyruvate from pyruvate. This prevents the plant from 'fixing' carbon dioxide into sugars in the dark (when there is no light to provide energy via photosynthesis). The regulatory protein, PDRP, can perform this vital function because it is dependent upon the presence of its substrate, ADP, for activity. This accumulates in the dark as a direct result of photosynthesis shutting down. We are interested in the details of the way in which the regulatory protein uses this molecule to inactivate PPDK. We will develop new ways to study this process and make mimics of the target which will help us further unravel the precise manner in which it occurs. The target protein is inactivated because a phosphate group is transferred from the substrate ADP to the protein, thereby blocking the part of PPDK required to perform its normal function. It is worth noting that using this particular molecule, adenine diphosphate (ADP), to perform this reaction is highly unusual, normally adenine triphosphate (ATP) is used instead.

Fixation of carbon dioxide in C4 photosynthetic plants is dark/light regulated via shut down of the C4 acid shunt between the leaf mesophyll cells and the bundle-sheath cells. This pathway serves to shuttle CO2 between the two cell types via reaction with phosphoenolpyruvate (PEP) to generate C4 organic acids in the mesophyll. These are transported to the bundle sheath cells where decarboxylation yields free CO2 which is fixed permanently by Rubisco. In the mesophyll, conversion of pyruvate to PEP is catalysed by pyruvate,orthophosphate dikinase (PPDK) which is in turn regulated by PPDK regulatory protein (PDRP). In the dark, increasing ADP levels in the mesophyll chloroplast are linked to the ADP-dependent inactivation of PPDK by PDRP. In the light, a decrease in the ADP concentration allows a phosphate-dependent reactivation to occur. In this proposal we are seeking to apply contemporary chemical biology methods to this enzyme system to investigate the manner in which the unusual ADP-dependent kinase functions. We will generate new continuous assay systems for PDRP and identify model non-proteigenic substrates for the enzyme. We will then use these methods and compounds to perform a full biophysical and kinetic characterisation of both activities of this unique protein.