Astley HM, Parsley K, Aubry S, Chastain CJ, Burnell JN, Webb ME, Hibberd JM

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 ...

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encoding 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. Among twenty-one highly conserved amino acids in RP1, mutating fifteen reduced kinase and phosphotransferase activity, while mutating six residues had no impact on either activity. We found no mutants in which only one activity was abolished. However, in some chimaeric fusions comprising the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity unaffected. These findings are consistent with both RP1 and RP2 reversibly modulating the activity of PPDK, and possessing one bifunctional active site or two separate sites in close proximity.

Date: Aug. 24, 2011

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