Structural And Functional Studies Of Maize Pyruvate Orthophosphate Dikinase Regulatory Protein (PDRP)

In nature,higher plants can be divided into three categories:C3,C4,and CAM(crassulacean acid metabolism),according to the different types of photosynthesis.In C4 plants,the most important rate-limiting C4 cycle enzyme is pyruvate,orthophosphate dikinase(PPDK),which catalyzes the reversible phosphorylation of pyruvate to phosphoenolpyruvate(PEP).As the primary CO2 acceptor,PEP plays an important role in the photosynthesis.PPDK is strictly subject to light-mediated regulation by reversible phosphorylation of Thr527 residue in maize.This process is accomplished by a single,bifunctional protein named pyruvate,orthophosphate dikinase regulatory protein(PDRP).In the dark,PDRP phosphorylates the threonine residue of PPDK in an ADP-dependent pathway,leading to the inactivation of PPDK;while in the light,PDRP dephosphorylates the phospho-threonine residue of PPDK in an inorganic phosphate(Pi)-dependent pathway,leading to the activation of PPDK.However,the molecular mechanism by which PDRP carries out its dual,competing activities,as well as the special structure of ADP-dependent kinase and Pi-dependent pyrophosphorylase,remain obscure.To solve these opened questions,in-depth studies were conducted in this study by means of structural biology combined with biochemistry and molecular biology.Through extensive screening of truncated variants,the protein crystal of maize PDRP42-426 was obtained.The structure of PDRP42-426·AMP complex was solved using single-wavelength anomalous diffraction.The whole structure forms a compact homo-dimer,besides the hydrogen bonds and salt bridge network,there are four disulfide bonds,important for the dimerization of PDRP.Each PDRP protomer contains two clearly separated N-terminal domain(NTD)and C-terminal domain(CTD)connected by a linker loop.Interestingly,both of the NTD and CTD have the same protein fold,which is a central four-parallel stranded ?-sheet packed against by three to four a-helices,forming a sandwich structure.CTD contains the conserved phosphate binding loop(P-loop)domain for binding nucleotides.The DALI consequences for NTD are protein receptors,showing that the function of NTD may be an allosteric effector which acts in vivo to modify the activity of the PDRP enzyme,or represents a module of protein-protein interaction domain with PPDK.DALI searches of CTD and whole PDRP resemble each other,share significant similarity with nucleoside monophosphate(NMP)kinases.By structural alignment between CTD and typical candidate of NMP kinase,we know the elaborate interaction between nucleotide and PDRP,in which the residues of P-loop and lid-like domain coordinate the ADP and magnesium.Then the in vitro kinase and pyrophosphorylase experiments show that the CTD alone performs similar enzymatic activities compared to the full-length PDRP,and the mutations at P-loop brought about a loss of nearly 100%of the enzymatic activities.Combined the structural analysis with in vitro kinase and pyrophosphorylase experiments,it is evident that both of the kinase and pyrophosphorylase active sites of PDRP locate in the CTD.Structural comparison between PDRP and HprK/Ps,belonging to P-loop nucleotide-binding protein kinases superfamily,shows that PDRP uses one shared active site for both kinase and pyrophosphorylase activities,and reveals the catalytic mechanism of PDRP:the P-loop for binding the substrates ADP and inorganic phosphate,residues Lys274 and Lys299 for neutralizing the negative charge,and residue Asp277 for protonating or deprotonating the target threonine residue of PPDK to promote nucleophilic attack and finish the pyrophosphorylase or kinase activity.The structure-guided mutagenesis shows that the mutants of key residues in the enzymatic active site abrogated both of the kinase and pyrophosphorylase activities of PDRP,further confirming the catalytic mechanism.In summary,this study provides a structural basis for understanding the catalytic mechanism of PDRP,and provides new scientific evidence to address the evolution relationship with other bifunctional protein kinase-phosphatases.Moreover,the unique structure feature of the NTD may be exploited for the development of selective activators or inhibitors to regulate the interaction between PDRP and PPDK,thus regulating the photosynthesis efficiency.