title: The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both bifunctional and interact with the catalytic and nucleotide-binding domains of pyruvate, orthophosphate dikinase.
abstract: Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in C(4)  photosynthesis and is also found in C(3)  plants. It is post-translationally modified by the PPDK regulatory protein (RP) that possesses both kinase and phosphotransferase activities. Phosphorylation and dephosphorylation of PPDK lead to inactivation and activation respectively. Arabidopsis thaliana contains two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of RP, and although RP1 has both kinase and phosphotransferase activities, to date RP2 has only been shown to act as a kinase. Here we demonstrate that RP2 is able to catalyse the dephosphorylation of PPDK, although at a slower rate than RP1 under the conditions of our assay. From yeast two-hybrid analysis we propose that RP1 binds to the central catalytic domain of PPDK, and that additional regions towards the carboxy and amino termini are required for a stable interaction between RP2 and PPDK. For 21 highly conserved amino acids in RP1, mutation of 15 of these reduced kinase and phosphotransferase activity, while mutation of six residues had no impact on either activity. We found no mutant in which only one activity was abolished. However, in some chimaeric fusions that comprised the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity remained unaffected. These findings are consistent with the findings that both RP1 and RP2 modulate reversibly the activity of PPDK, and possess one bifunctional active site or two separate sites in close proximity.
authors: Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, UK.
authors: Holly M
authors: HM
authors: Astley
authors: Kate
authors: K
authors: Parsley
authors: Sylvain
authors: S
authors: Aubry
authors: Chris J
authors: CJ
authors: Chastain
authors: Jim N
authors: JN
authors: Burnell
authors: Michael E
authors: ME
authors: Webb
authors: Julian M
authors: JM
authors: Hibberd
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011772:Pyruvate, Orthophosphate Dikinase / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; C528952:RP1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases; C528953:RP2 protein, Arabidopsis; D011772:Pyruvate, Orthophosphate Dikinase