Molecular characterisation of an ADP-dependent regulatory protein

Lead Research Organisation: University of Leeds
Department Name: Sch of Chemistry

Abstract

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.

Technical Summary

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.

Publications

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McAllister TE (2012) Triazole phosphohistidine analogues compatible with the Fmoc-strategy. in Organic & biomolecular chemistry

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McAllister TE (2013) Prospects for stable analogues of phosphohistidine. in Biochemical Society transactions

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McAllister TE (2011) Fmoc-chemistry of a stable phosphohistidine analogue. in Chemical communications (Cambridge, England)

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Williamson DJ (2012) Efficient N-terminal labeling of proteins by use of sortase. in Angewandte Chemie (International ed. in English)

 
Description In this project we studied the mechanism of a plant enzyme which is central to how some crop plants can survive in hot and arid conditions. These plants have a unique way of converting CO2 into sugars through photosynthesis and the enzyme studied in this project, PDRP, links this processto whether the plant is illuminated or not. The precise chemical mechanism used by this regulatory mechanism is unprecedented throughout known systems

In this project, we optimised protocols to isolate the relevant enzymes from plants as well as the homologous enzymes from bacteria. This is challenging and our studies represent the first high-yielding purification to homogeneity of these proteins. We then investigated the ligand-binding properties of these enzymes, and generated peptide ligands for the enzymes using our reported synthesis of phosphohistidine analogues. These studies suggest an ordered binding mechanism for the enzyme, in which the cofactor substrate, ADP, binds before interaction with the protein substrate.

Site-directed mutagenesis studies on both the plant and bacterial enzymes enabled us to identify regions of the protein sequence required for binding to the ADP substrate, studies to identify conserved residues interacting with the protein substrate are ongoing. Unfortunately we were not able to crystallise the enzymes to obtain a 3-dimensional model of the system.
Exploitation Route At the present time, we have estabilised the first conditions to robustly overexpress the enzymes and have generated peptide substrates which can be used in future enzymatic characterisation. These reagents can be used in tandem with in vivo studies to fully understand how the system is regulated.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description No known direct impact to date.
 
Description Marie Sklodowska-Curie Individual Fellowship
Amount £255,000 (GBP)
Funding ID 657978 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 08/2015 
End 07/2017
 
Description Salters festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Increased interest in chemistry for school students

Increased application to science courses
Year(s) Of Engagement Activity 2012,2013
 
Description Sixth form open labs 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Increased interest and understanding of research chemistry

Increased applications to STEM courses
Year(s) Of Engagement Activity 2008,2010,2012