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BBRC Biochemical and Biophysical Research Communications 345 (2006) 675–680 www.elsevier.com/locate/ybbrc Cloning and expression of maize-leaf pyruvate, Pi dikinase regulatory protein gene Jim N. Burnell a a,* , Chris J. Chastain b Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Qld 4811, Australia b Department of Biosciences, Minnesota State University-Moorhead, Moorhead, MN 56563, USA Received 5 April 2006 Available online 4 May 2006 Abstract Pyruvate, orthophosphate dikinase (PPDK; E.C. 2.7.9.1) catalyzes the synthesis of the primary inorganic carbon acceptor, phosphoenolpyruvate in the C4 photosynthetic pathway and is reversibly regulated by light. PPDK regulatory protein (RP), a bifunctional serine/ threonine kinase-phosphatase, catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PPDK. Attempts to clone the RP have to date proven unsuccessful. A bioinformatics approach was taken to identify the nucleotide and amino acid sequence of the protein. Based on previously established characteristics including molecular mass, known inter- and intracellular location, functionality, and low level of expression, available databases were interrogated to ultimately identify a single candidate gene. In this paper, we describe the nucleotide and deduced amino acid sequence of this gene and establish its identity as maize PPDK RP by in vitro analysis of its catalytic properties via the cloning and expression of the recombinant protein. Crown Copyright 2006 Published by Elsevier Inc. All rights reserved. Keywords: PPDK; PPDK regulatory protein; RP; C4 photosynthesis; Protein phosphorylation/dephosphorylation; Maize; Zea mays Pyruvate, orthophosphate dikinase (PPDK), an enzyme found in plants and certain microorganisms, catalyzes the reversible phosphorylation of pyruvate to phosphoenolpyruvate (PEP) [1]: ADP AMP RP PPDK-Thr PPDK-ThrP PPDK Pyr þ ATP þ Pi $ PEP þ AMP þ PPi In C4 plants it serves to regenerate the primary CO2 acceptor PEP [1], while its role in C3 plants is not fully understood [2]. As one of two potentially rate-limiting enzymes of the C4 pathway [1,3], PPDK activity is strictly light regulated post-translationally by reversible phosphorylation of an active-site Thr residue (Thr456 in maize). A single, bifunctional protein kinase/phosphatase named the PPDK regulatory protein (RP) catalyzes this regulatory phosphorylation/dephosphorylation cycle [4,5]: * Corresponding author. Fax: +61 7 47816078. E-mail address: [email protected] (J.N. Burnell). Active Inactive PPi Pi It is a highly unusual regulatory enzyme in at least three important respects. First, it is bifunctional in that it catalyzes both PPDK inactivation (phosphorylation) and activation (dephosphorylation). This is quite rare because most regulatory phosphorylation cycles have separate kinase and phosphatase enzymes [6,7]. Second, it uses ADP instead of ATP as the phosphoryl donor. Third, it employs a Pi-dependent, inorganic pyrophosphate (PPi)-forming phosphorolytic dephosphorylation mechanism, as opposed to simple hydrolysis as in most protein phosphatases [8]. 0006-291X/$ - see front matter Crown Copyright 2006 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.04.150 676 J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680 Although the protein has been partially purified a number of times (e.g., see [4,8,9]) the enzyme has defied all attempts to determine its molecular sequence. Specifically, the enzyme is of such low abundance in the final fractions that obtaining authentic peptide sequences from onedimensional SDS–PAGE gels has proved elusive. In what appeared to be a breakthrough for its cloning, Burnell, using a similar purification protocol, was able to obtain a preparation of RP polypeptide that generated antibodies capable of inhibiting the RP inactivation reaction in assays containing chloroplasts as a source of both RP and PPDK (unpublished results). When these antibodies were used to screen a maize leaf cDNA expression library for candidate RP clones, an RP-encoding clone could not be recovered. Thus, without authentic peptide sequence or highly specific antibodies, cloning of RP using conventional approaches was essentially precluded. More recently, advanced approaches for cloning RP, such as the bacterial two-hybrid system, were attempted with PPDK serving as the bait protein in a bacterial cell line transformed with a maize leaf cDNA expression library. However, thorough screening of two-hybrid libraries consistently failed to identify clones that encoded proteins capable of catalyzing ADP-dependent inactivation of PPDK. Nor did the use of site-directed mutagenesis of the bait PPDK regulatory threonine and/or the catalytic-histidine residues (e.g., in attempts to increase the interaction or dwell time of RP with PPDK) reveal a positive clone (Tems and Burnell, unpublished results). It was with the advent of ever-expanding database sequences for a range of vascular plant species that we finally opted for a bioinformatics approach as the most viable means for cloning the RP gene. We sought to do this by identifying those sequences that were consistent with the known properties of the RP. These included plant proteins with (i) an estimated molecular mass of between 45 and 48 kDa, (ii) a chloroplast transit peptide, (iii) a threonine/ serine protein kinase domain, (iv) a phosphate/pyrophosphate binding domain, and (v) a relatively high proportion of hydrophobic residues (as based on its salting-out profile). Moreover, we expected the gene to be classified as having ‘‘unknown function’’ based on the absence of any other eukaryotic protein of like catalytic function. Additionally, since RP activity has now been demonstrated in C3 plant chloroplasts [2] we also expected C3 plant RP homologs to be present in C3 plant databases. From these properties, a number of likely candidates were identified in maize and then cross-referenced with the rice and Arabidopsis genome databases. Augmenting our database search was a recently published proteomics study that compared expression levels of soluble, stromal polypeptides in isolated maize-leaf bundle sheath and mesophyll cell chloroplasts [10]. This information facilitated the identification of the most likely RP gene candidate within our search (GenBank Accession Nos. AY106112 and AY106855). We then generated a PCR product corresponding to the respective ORF sequence of this gene and subsequently utilized it as a probe for screening a maize-leaf cDNA library. Positive clones were recovered as excised plasmids and the cDNA insert subsequently subcloned into an Escherichia coli expression vector for producing the encoded protein recombinantly. Reported here are experiments performed with this recombinant protein that confirm its identity as the maize PPDK bifunctional regulatory protein. Materials and methods A maize leaf cDNA library constructed in kZAP (Stratagene) was screened with a radioactively labeled 491 bp PCR fragment generated using the cDNA library as template and two internal primers identified using MacVector. Sequences of the two primers were 5 0 CCACCTCTTCTCCTTGATTGACG-3 0 (forward primer) and 5 0 CAGTCCAAAAACCTTGTCCTGG-3 0 (reverse primer). About 25 ng DNA was radiolabeled using a Decaprime Labeling Kit (Ambion) and [a-32P]dATP. Positive plaques were isolated and rescreened and pure plaques isolated. Plasmid DNA was obtained by in vivo excision using Helper phage according to the manufacturer’s instructions (Stratagene). The nucleotide sequence of the plasmid DNA was determined (Macrogen, Korea). An insert containing the coding region of full length DNA was obtained by digesting plasmid DNA with NheI, a restriction enzyme that digests the plasmid insert six bases upstream of the initiating ATG start codon, and KpnI which digests within the polylinker of pBluescript, and ligated into pROExC digested with SpeI and KpnI. Following heat transformation of competent NM522 cells in the presence of ligated DNA, cells were grown overnight on LB carbenicillin (LBC) plates and colonies used to inoculate 5 ml LBC cultures. After overnight shaking a sample of each culture was removed and used to prepare a glycerol stock. Plasmid DNA was isolated and digested with a variety of restriction enzymes to confirm the presence of the insert in the vector. Glycerol stocks of cultures containing cells with successfully cloned inserts were used to inoculate 5 ml cultures which were then used to inoculate 500 ml cultures (in 2 L baffled flasks) and shaken. After 3 h IPTG was added to a final concentration of 0.5 mM and the cultures grown overnight at 25 C. Cells were harvested by centrifugation, washed with extraction buffer containing 25 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 1 mM Pi, and 0.5 mM b-mercaptoethanol, the cells resuspended in 5 ml of buffer and frozen at 80 C. Cells were sonicated (10 s bursts for 2 min at 80% output—Branson Sonifier 450) and the cell debris removed by centrifugation at 40,000g for 30 min. The crude supernatant was decanted and applied to a 5 ml nickel–NTA affinity column (Pharmacia) and washed with buffer until the OD280nm decreased below 0.5. The column was then washed sequentially with buffer containing 5 mM imidazole until the OD280nm decreased below 0.5 and protein eluted from the column with a solution containing 20 mM imidazole in buffer. Those fractions containing eluted protein were tested for ADP-dependent PPDK inactivation activity and Pi-dependent activation. Inactivation assay. RP catalyzed inactivation of active (dephospho) maize C4 recombinant PPDK (EC 2.7.9.1; GenBank Accession No. J03901) was performed using a previously published procedure including the following modifications [8]. Pre-activated (pre-warmed at 25 C for 60 min) Ni–NTA purified recombinant maize C4 PPDK [11] (0.15 U) was added to a reaction mixture containing 0.1 ml column buffer and 25 ll of recombinant RP containing column eluate in a final volume of 0.2 ml. Inactivation reactions were initiated by the addition of 20 ll of 20 mM ADP/1 mM ATP. At various intervals, 20 ll samples were withdrawn and assayed for PPDK activity using a spectrophotometric assay as previously described [12]. After 10 min incubation, 50 ll of the inactivation assay reaction was added to an equal volume of SDS–PAGE buffer (125 mM Tris–HCl, pH 6.8, 12% w/v SDS, 10% v/v glycerol, 0.01% w/v bromophenol blue, and 0.5% b-mercaptoethanol). The quenched samples were then heated at 95 C for 5 min prior to loading aliquots on SDS–PAGE gels as described below. Pi-dependent PPDK reactivation assays. Purified recombinant PPDK and RP were incubated with 2 mM ADP/0.1 mM ATP at 25 C. After J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680 10 min the whole incubation mixture was passed through a column (0.4 · 6 cm) of Sephadex G25 to remove the nucleotides [5]. Inorganic phosphate was added to the column eluate to a final concentration of 1 mM and 25 ll samples removed and spectrophotometrically assayed for PPDK activity [12]. SDS–PAGE and Western blot analysis. Proteins present in the terminated reaction mixtures were separated by SDS–PAGE on 10% polyacrylamide gels [13] and the separated proteins transferred to HybondP (Amersham) membrane. The resulting blots were probed with either one of two primary maize PPDK rabbit polyclonal antibodies. For specific detection of RP catalyzed Thr456-phosphorylated PPDK, affinity-purified polyclonal antibodies raised against a synthetic phosphopeptide conjugate corresponding to the phosphorylation domain of maize C4 PPDK (encompassing residues 445–464 [AVGILTERGGMpTSHAAVVAR] [14,15]) were used. For detection of total PPDK (phospho- and dephospho-) polyclonal antibodies raised against the recombinant maize C4 PPDK monomer were used. For following His-tagged recombinant RP during purification, anti-Tetra-His antibodies (Qiagen) were also used. Proteins on Western blots were visualized using a secondary antibody conjugated to either horseradish peroxidase or alkaline phosphatase. Results and discussion Identification of a potential RP candidate using in silico bioinformatics screening Previous in vitro studies of partially purified maize leaf RP demonstrated that RP was functionally a threonine/ serine kinase in that Ser but not Tyr could substitute for its target Thr456 residue [2,14,15] yet utilizing ADP as the phosphoryl donor [8]. In previous reports where the protein had been purified [4,8,9] the protein was shown to precipitate at low ammonium sulfate concentrations suggesting the protein is relatively hydrophobic. Furthermore, maize RP has also been demonstrated to be co-localized along with its target protein, PPDK, to the chloroplast stroma of C4 leaf mesophyll cells [16]. Thus, along with PPDK as a nuclear encoded protein, its gene must also encode a chloroplast transit peptide [17]. Additional insights into RP gained from previous purification studies include an estimated molecular mass of 45–48 kDa and that it is typically present in maize leaves as a very low abundant protein [8,9]. Lastly, since no other known eukaryotic protein possesses the bifunctional catalytic properties of RP, we expected any RP candidate genes to be classified as ‘‘unknown’’. These collective characteristics were then utilized for interrogating the maize databases for candidate RP genes. From this search, a list in excess of about 50 maize proteins was compiled and these sequences cross-referenced with related sequences in the rice and Arabidopsis databases. As this search was nearing completion, a comparative proteomics study was published that profiled differences in soluble chloroplast polypeptide content between maize leaf bundle sheath and mesophyll cell-types [10]. We noted that a partial peptide sequence corresponding to an unknown protein [ZmGI Accession No. TC220929] was of apparent low abundance and expressed more or less exclusively in mesophyll chloroplasts (bundle sheath: mesophyll ratio of 0.04). More extensive sequence information indicated the 677 corresponding pre-protein had a molecular mass of 45.9 kDa including a predicted (ChloroP) [18] chloroplast transit peptide of 42 amino acids, leaving a mature protein with a molecular mass of 41.6 kDa. We then proceeded to clone the corresponding cDNA for the ZmGI TC220929 gene (GenBank Accession No. AY106855) by screening a maize leaf cDNA library with a cloned 491 bp PCR fragment complementary to sequences internal to the respective AY106855 ORF. Screening of the library with this 491 bp 32P-labelled probe detected about 50 positive plaques out of 25,000 plaques. In vivo excision of plasmid DNA from the lambda clones produced ‘‘two families’’ of clones that differed in sequence length within the 3 0 -non-coding region (as confirmed by DNA sequencing). Examination of the amino acid sequences of these two maize leaf RP clones with SignalP 3.0 [19] and ChloroP 1.1 [18] suggested a common chloroplast transit sequence with a cleavage site between amino acid residues 42 and 43, leaving a mature protein of 384 amino acid residues with a molecular mass of about 41.5 kDa. A more detailed examination of the full-length protein with PROSITE [20] identified (i) a protein kinase C phosphorylation site, and (ii) an ATP/GTP-binding motif A (P-loop) between residues 293 and 300, both consistent with the functional identity of the protein as a serine/threonine protein kinase. Interestingly, a protein BLAST [21] search across all phyla revealed that this protein shares a large, centrally oriented conserved domain called DUF299 (Domain of Unknown Function) with a relatively large number of PPDK-possessing bacterial species, in addition to rice and Arabidopsis. For ascertaining the identity of this gene as RP, we needed to show in vitro that the recombinantly produced protein had the unique catalytic properties of maize leaf RP. That is, Thr456-specific, ADP-dependent phosphorylation of the PPDK catalytic intermediate (PPDKHis458-P) and Pi-dependent dephosphorylation of the same ThrP residue. To accomplish this, the ORF of the shorter clone was subcloned into an E. coli expression vector that encoded a 6· His-tag N-terminal to the translational start codon of the native ORF. The expressed pre-protein was purified to >80% purity by Ni-affinity column chromatography (Fig. 1, lane 4). This RP active fraction was eluted with 20 mM imidazole with an apparent molecular mass of 40 kDa. This preparation was used in subsequent ADP-dependent PPDK inactivation assays (Table 1) and for the Western blot-based in vitro phosphorylation/dephosphorylation assays (Fig. 2B). In the in vitro test of the recombinant protein to inactivate PPDK in an ADP-dependent manner, we clearly show robust inactivation of active, recombinant maize C4 PPDK (Table 1). Importantly, not only was the inactivation shown to be ADP-dependent, but that pyruvate could inhibit PPDK inactivation by this protein further underscores its identity as RP. This specificity is inferred because only the catalytic intermediate form of PPDK (phosphoHis458) can serve as the phosphorylation substrate of 678 J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680 MW 1 kDa 110 77 2 3 4 5 6 A MW 1 2 3 4 5 6 44 29 B MW Fig. 1. Coomassie blue-stained SDS–PAGE of bacterially expressed RP purified by nickel-affinity column chromatography. RP, expressed in 1 L of bacterial culture, was purified on a 5 ml nickel affinity column. The column was loaded with crude extract (lane 2), the column washed with column buffer (lane 1), 5 mM imidazole in column buffer (lane 3), 20 mM imidazole in column buffer (lane 4), and 200 mM imidazole in column buffer (lane 5). Purified maize recombinant PPDK was loaded in lane 6. MW; prestained molecular weight markers. RP, not simply the enzyme with an unphosphorylated Thr456 residue [14,22,23]. Thus, by inclusion of the substrate pyruvate in the inactivation reaction the His458 phosphate is immediately transferred to pyruvate catalytically (in forming PEP) thus converting PPDK back to the non-phosphorylated, non-RP substrate His458 state (Table 1 and Fig. 2). Western blots of quenched aliquots of the PPDK inactivation reactions probed with maize C4 PPDK phosphopeptide antibodies provided unequivocal immunological confirmation that the recombinant protein catalyzed site-specific (Thr456) phosphorylation of PPDK. A companion blot probed with maize C4 PPDK (phospho + dephospho) antibodies indicated the presence of equal amounts of maize C4 recombinant PPDK in each of the reaction mixtures, indicating the phospho-PPDK signals in Fig. 2B can be viewed as quantitative (Fig. 2A). For demonstrating that the cloned recombinant protein could also catalyze the opposing RP reaction, that is, Pi-dependent dephosphorylation of Thr456-P-PPDK, the end-point reaction mixture from the PPDK inactivation/phosphorylation assay was desalted by Sephadex G25 column chromatography and re-incubated for 10 min in the presence of 1 mM Pi. Aliquots of this reaction were 1 2 3 4 5 6 Fig. 2. Inactivation of PPDK by bacterially expressed RP. Inactivation reactions were run and terminated reaction mixtures subjected to SDS– PAGE, proteins transferred to Hybond-P membrane, and proteins located using. (A) Rabbit anti-PPDK antibodies. (B) Rabbit anti- Thr456-P phosphopeptide antibodies. Lane 1, complete reaction; lane 2, minus ADP/ATP; lane 3, minus RP; lane 4, PPDK inactivated in a complete reaction, the reaction mixture run through a Sephadex-G25 column, and activation continued for 10 min with 1 mM Pi; lane 5, no PPDK; lane 6, complete reaction plus 2 mM pyruvate. analyzed for phospho-PPDK content via Western blots probed with maize C4 PPDK phosphopeptide antibody as before. As evident from the signal intensity of the immunoreactive bands in lanes 1 and 4, Fig. 2B, the recombinant RP was able to catalyze a decrease in the level of phosphoPPDK (versus control). This Pi-dependent decrease in the amount of phosphorylated (inactivated) PPDK is consistent with the corresponding level of increase in PPDK activity as determined in a parallel PPDK spectrophotometric-based activation assay of the same desalted reaction mixture (64% of the original PPDK activity, Table 1). Further confirmation that the recombinant protein possessed RP-like, Pi-dependent PPDK activation property was provided by the demonstration that adding inorganic pyrophosphatase to the PPDK activation reaction significantly increased the rate and final extent of activation Table 1 ADP/ATP-dependent inactivation of purified PPDK by bacterially-expressed RP Reaction no. Reaction mixtures PPDK activity at time zero (%) PPDK activity after 5 min (%) PPDK activity after 10 min (%) 1 2 3 4a 5 6 7 Complete Minus ADP/ATP Minus RP Complete Plus 2 mM pyruvate Minus PPDK Plus 2 mM pyruvate 100 100 100 Not assayed 100 0 100 70 104 93 Not assayed 97 0 97 47 108 105 Not assayed 99 0 99 PPDK and ADP-dependent inactivation activities were assayed as described in Materials and methods. a PPDK activity of reaction 4 was not determined during inactivation. Following the addition of ADP/ATP, reaction 4 was incubated at 25 C for 10 min and then subjected to Sephadex G25 column chromatography. The desalted reaction mixture was then incubated at 25 C for 10 min with 1 mM Pi and PPDK activity determined to be 64% of the time zero activity of reaction 1. Remaining reaction mixtures were subjected to SDS–PAGE and Western blot analysis (Fig. 2). J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680 of inactivated PPDK (result not shown). This is consistent with the RP dephosphorylation mechanism as a Pi-dependent PPi forming phosphorolytic cleavage, as opposed to a simple hydrolysis as with most protein phosphatases. Thus, removal of PPi by pyrophosphatase should thermodynamically ‘‘pull’’ the reaction forward [4], as is demonstrated here. As mentioned above, PPDK is not only present in plants but it is also present in a select variety of bacteria, most of which are facultative anaerobes. A striking observation is that these same PPDK-containing bacteria also encode the DUF299 gene, the same large domain of sequence that comprises most of the maize RP polypeptide (see Fig. 3). Since most of the PPDK-DUF299 bacterial species are facultative anaerobes, it is notable that PPDK provides a far different function in these bacteria than it does in C4 plants. For example, it has been proposed that the enzyme supplants pyruvate kinase in the glycolytic pathway for anaerobic synthesis of cellular ATP [24]. It is possible that a 10 Maize RP Sa At Na St Gm Lm E A E R D A A A G M E D Q M E N K K S F M Q R M V A K M N G S M M E N P E A Maize RP Sa At Na St Gm Lm E Q Q R E S E V V V I V A T V I F I M I L L . K E D G D E E . Q E L E A D E . A A I I A V A A A S D A R L K K Q K A Q K A E K E N Q E A Maize RP Sa At Na St Gm Lm R L R K K Q K . S G R G L N Q F E R R E E D . P P V P P P P P S G G G G G G G R I A R R L N N L Q Q I L M G R H H H Y R . R R A M R E R R Maize RP Sa At Na St Gm Lm K K R R R K Q . G N G G R G N G Y I I Y Y Y W Y K K K K K K K K V V T V V V V V A I A A A A A A N N N N N N N N V I I I L V V . P P P P P P P P Maize RP Sa At Na St Gm Lm E S D D S D S . M L R N M Y E D E A E E E K E H R T Q R K R . V I I V I I I I R L M T L E L Q E E R Q D E . E E E E E E E E L T L V L L L L K L F L P R V I I L L I I I I Y F H H F Y Y . I I L L V L V . V I I L V F I . S S S S S S S S D D D D D D D D G S S S S A A S T L T T V T I T A V G G T G G G L F I L L I I . V M V V I V V V L M L L V I V . Y F Y Y Y Y Q Y T S T T T T T T L L I L I L L L A V V V I V V V D D D N V D Q D S D N D D N D D E N A E D T S D D D A D E N D Y Y Y Y Y Y Y Y F F F F F H F F Q K A A R K D R R R R R R R R I V I V I I I I D E E D E E A E V V V V V V V V M P P V P K P P G E N E E G E E V V V S V I I V A P P P S D P P L V L P L P I . P P P P P P P P K K E S K A A S E S M E E E E H Y Y F Y Y Y Y A A A A A C G A N E R R E R Y Q E A R S Q D I V L M V L T . F M C F F Y F F V K A A K R K . Q N R D K R K . N L N N V N L N 70 G T P P P P D P T T T T T T T T A A L L A V A A E K M E D E Q . H A S M L R H S I A I V V I . V A G A T L I . N K R K R R R R A A A A A A A A P V Q A P A A . S A Q E E E K M L L T I L L L A A A R R A A A E S E E K Q E E A Y F R E L Y T A L L L L A L K Q D E Q R T K K L Q R D N A R R R L E F A S A A A A A A I I L I V V I I D E N Q E D E . F F F F F F F F T A A T A T A A I V M I V V V V K K D A K K K K Q Y H H Y H Y D D D D D D D D D D D D D D D D L L L L L L L L F R Y F F F F F E M A S L K Q I I A L V V I . N D T R P D P Q S R R P Q A D R P P H N E K R L L K K R . V I I V V I I I F I V V V V I V W Q - W C W W C W C W P P P P A L Q P V I L V V V V V I I I I I I I I A N D D D D H D V V V V V V V V T S T S T T E T G D R R N K D 40 L L S L L L T L G S A A S S A . Q Q Q Q Q Q Q Q F F F F W F F F E P H D G R S N G L C C L L A A C C C C V A C C D E H A D E V . F S A A M A Q W E I L W H N G H G G D G N G V Y V L I L I . P A P P P D P P C Y C S A A N T V V V V L V V D D N A D D D D G G G G G G C G V R Q V K Q K L D M G D D D D P P P W P P P P Q R E E R R R R N G T N G G G G F I Y W F L L . Y L D E L S L R Q D E R K D A A A A A A A A L L L L L L L L T T V T R L T T I N A T V I T . N S T S S D T . P V S P P P P P A D D E E H E . I H R R K R K . L L I L L L L L Q N S I H L A A A S S A S A A I I I I V V I I E E E E E E E E E E E E E E E E T T T T T S T T A A A A A A A A A T A A V A A A V I A A R E L . I I I V V I I I V L V A I L L . D D D D D D D D G Q Q A Q E Q Q C D S A R V K A D M D S E P V W P V V A A . N H V V E R D T F E R I I I H Q H H R H R H L C V F R R R L L L L M I L L R T E D G S H P N P A P S T T V I V M L L . V I I T L L T . E Q G D D F A A G I A G K A . I I F L I L V . A S Q Q S S E S S P P P S A L Y M M M K F I I I V I I I I D N R R D R R R D S N S D T D M Q Q M P R E D E K Q Q E S R R Q H S D A L L L L L I L L H I Y Q L F K I S L L L F T . G G G G G G G G V I A I A V L . A D P Q P A K P P S S G G I I I L V I A F I . V V V V V V V V L I I L L L L L A I I A I V V . G G G G G G G G V I I V V V V V S S S S S S S S R R R R R R R R T T T S S S T T K K K K K K K K T T T T T T T T P P P P P P P P L L T T V L L L S S S S S S S S I M I I M M S Y F Y Y Y Y F Y L L L L L L L L A A A A A A A A I V V V I I I I R R R R R R R R K K E R E S K . T V N N E V V R R R R R R R R D D A L L L I L L L K K G L K K K K T S T S T N S . G G G N G G G G F L G Q L Q L . D S F A R M D G S D P S S E R T G E D R A Q Q S A G T A G S . N N R A N S S Y Y Y Y Y Y Y Y G E A N E Q E I I L L I K I I L L R Y I L I . H K P N N C T D T R E R S S R N T R G K G R T V Q K C S M P R I S K R Y * Q V A K N L 110 S R T Q R D A G S S S S S S S P P S A E P S 180 230 L L T L I L I L I E A D R L L . 120 170 280 L L L I L I T L F F Y W F M H . 60 G H T G Q D N 220 270 K K R R K R K K 50 R R 160 210 G G G G G G G G D D 100 150 260 P G N G G P G G A C V A A A V A 90 200 250 V R K Q D N E 30 W E E E E E E E 140 190 I L V I L L L L G G G G G G G G 80 130 L L L M L L L L ‘‘prokaryotic’’ RP, as the DUF299 gene product, evolved in bacteria in order to regulate the ATP synthesizing function of PPDK. Since the DUF299 contains a GTP-binding domain it is reasonable to speculate that RP evolved in bacteria to down-regulate PPDK activity via an ATPdependent phosphorylation of the active-site Thr (also strictly conserved in bacterial PPDKs), as opposed to the ADP-dependent inactivation-phosphorylation mechanism that RP catalyzes in plants. In such a regulatory scheme, when cellular ATP begins to accumulate (e.g., at low energy demand) an ATP-dependent feedback inhibition of glycolysis via down-regulation of PPDK would ensure conservation of PEP during glycolytic ATP synthesis. It may be significant in this regard that maize RP catalyzes the ADP-dependent inactivation of PPDK from the nonsulfur purple photosynthetic bacterium Rhodospirillum rubrum [25]. In plants, PPDK inactivation is dependent on the PPDK being catalytically His-phosphorylated before it can be 20 A T H H V H I 679 Q D N N H H N . 240 290 A A T V S A S . 300 A A G A S G D G V K P I N F G Y H K Fig. 3. Amino acid sequence alignment of maize RP. The maize RP deduced amino acid sequence was aligned, using the ClustalW alignment program of the MacVector 8.0 software, with orthologs identified in Agrobacterium tumefaciens AAL41032.1 (At), Geobacter metallireducens ABB33596.1 (Gm), Listeria monocytogenes AAT04664.1 (Lm), Novosphingobium aromaticivorans ABD24566.1 (Na), Streptococcus agalactiae CAD47374.1 (Sa), and Symbiobacterium thermophilum BAD39570.1 (St). These bacterial species also contain orthologs of plant PPDK. The first 130 amino acids of the maize RP sequence have been removed. Regions of amino acid identity are indicated with shading and consensus sequence shown under the aligned sequences. 680 J.N. Burnell, C.J. Chastain / Biochemical and Biophysical Research Communications 345 (2006) 675–680 phosphorylated by the ADP-dependent RP-catalyzed reaction; the catalytic phosphate is derived from ATP [22]. It has also been reported that PPDK may be catalytically phosphorylated by pre-incubation with PEP [22]. Therefore, it is logical to suggest that if RP functions to regulate PPDK activity in bacteria (in the ATP-forming direction) and the substrate for inactivation is the catalytically phosphorylated form, it is likely that PEP is the immediate phosphate donor for catalytic phosphorylation. So it is possible that the ADPdependent inactivation of PPDK evolved in bacteria to regulate PPDK catalyzing the formation of ATP and pyruvate and that this regulatory mechanism has been adopted by plants that use PPDK in either direction (i.e., pyruvate- or PEP-forming). The fact that PPDK needs to be catalytically phosphorylated before it can be inactivated may have evolved to regulate PPDK in the ATP-forming direction (glycolytic direction) and the true substrate for catalytic phosphorylation is PEP. In contrast, in plants in which PPDK operates in the PEP-forming direction ATP provides the catalytic phosphate. And finally, the nucleotide substrate specificity of RP has altered from using ATP in bacteria to using ADP in plants as the function of PPDK altered. The role of PPDK and RP in microorganisms is currently under investigation and will be discussed elsewhere. Acknowledgment J.N.B. wishes to thank Timothy Sexton for technical assistance. References [1] G.E. Edwards, H. Nakamoto, J.N. Burnell, M.D. Hatch, Pyruvate,Pi dikinase and NADP-malate dehydrogenase in C4 photosynthesis: properties and mechanism of light/dark regulation, Annu. Rev. Plant Physiol. 36 (1985) 255–286. [2] C.J. Chastain, R. Chollet, Regulation of pyruvate, orthophosphate dikinase by ADP-/Pi-dependent reversible phosphorylation in C3 and C4 plants, Plant Physiol. Biochem. 41 (2003) 523–532. [3] R.T. Furbank, J.A. Chitty, C.L.D. Jenkins, W.C. Taylor, S.J. Trevanion, S.V. Caemmerer, A.R. Ashton, Genetic manipulation of key photosynthetic enzymes in the C4 plant Flaveria bidentis, Aust. J. Plant Physiol. 24 (1997) 477–485. [4] J.N. Burnell, M.D. Hatch, Regulation of C4 photosynthesis: purification and properties of the protein catalyzing ADP-mediated inactivation and Pi-mediated activation of pyruvate, Pi dikinase, Arch. Biochem. Biophys. 237 (1985) 490–503. [5] J.N. Burnell, M.D. Hatch, Dark/light regulation of pyruvate,Pi dikinase in C4 plants: evidence that the same protein catalyses activation and inactivation, Biochem. Biophys. Res. Commun. 111 (1983) 288–293. [6] S.K. Hanks, T. Hunter, The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification, FASEB J. 9 (1995) 576–596. [7] R.D. Smith, J.C. Walker, Plant protein phosphatases, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996) 101–125. [8] A.R. Ashton, J.N. Burnell, M.D. Hatch, Regulation of C4 photosynthesis: inactivation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and activation by phosphorolysis, Arch. Biochem. Biophys. 230 (1984) 492–503. [9] C.M. Smith, S.M.G. Duff, R. Chollet, Partial purification and characterization of maize-leaf pyruvate,orthophosphate dikinase regulatory protein: a low abundance, mesophyll-chloroplast stromal protein, Arch. Biochem. Biophys. 308 (1994) 200–206. [10] W. Majeran, Y. Cai, K.J. van Wijk, Functional differentiation of bundle sheath and mesophyll maize chloroplasts determined by comparative proteomics, Plant Cell 17 (2005) 3111–3140. [11] J.R. Doyle, J.N. Burnell, D.S. Haines, L.E. Llewellyn, C.A. Motti, D.M. Tapiolas, A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides, J. Mol. Screening (2005) 67–75. [12] A.R. Ashton, J.N. Burnell, R.T. Furbank, C.L.D. Jenkins M.D. Hatch, The enzymes in C4 photosynthesis, in: P.J. Lea (Ed.), Methods in Plant Biochemistry, Academic Press, vol. 7A, 39–72. [13] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T7, Nature 227 (1970) 680–685. [14] C.J. Chastain, M. Botschner, G.E. Harrington, B.J. Thompson, S.E. Mills, G. Sarath, R. Chollet, Further analysis of maize C4-pyruvate, orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues, Arch. Biochem. Biophys. 375 (2000) 165–170. [15] C.J. Chastain, M.E. Lee, M.A. Moorman, P. Shameekumar, R. Chollet, Site-directed mutagenesis of maize recombinant C4-pyruvate, orthophosphate dikinase at the phosphorylatable target threonine residue, FEBS Lett. 413 (1997) 169–173. [16] M.D. Hatch, J.N. Burnell, Regulation of C4 photosynthesis: stability and other characteristics of the ADP-ATP-dependent inactivation of pyruvate, Pi dikinase in maize chloroplast extracts, Aust. J. Plant Physiol. 10 (1983) 179–186. [17] D.R. Hague, M. Uhler, P.D. Collins, Cloning of the cDNA for pyruvate, Pi dikinase from maize leaves, Nucleic Acids Res. 14 (1983) 4853–4865. [18] O. Emanuelsson, H. Nielsen, G. von Heijne, ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites, Protein Sci. 8 (1999) 978–984. [19] J. Dyrløv Bendtsen, H. Nielsen, G. von Heijne, S. Brunak, Improved prediction of signal peptides: SignalP 3.0, J. Mol. Biol. 340 (2004) 783–795. [20] A. Bairoch, P. Bucher, K. Hofmann, The PROSITE database, its status in 1997, Nucleic Acids Res. 25 (1997) 217–221. [21] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. [22] J.N. Burnell, M.D. Hatch, Regulation of C4 photosynthesis: catalytic phosphorylation as a pre-requisite for ADP-mediated inactivation of pyruvate, Pi dikinase, Biochem. Biophys. Res. Commun. 118 (1984) 65–72. [23] J.N. Burnell, M.D. Hatch, Regulation of C4 photosynthesis: identification of a catalytically important histidine residue and its role in the regulation of pyruvate, Pi dikinase, Arch. Biochem. Biophys. 231 (1984) 175–182. [24] E. Saavedra-Lira, L. Ramirez-Silva, R. Perez-Montford, Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica, Biochim. Biophys. Acta 1382 (1998) 47–54. [25] R.J.A. Budde, S.M. Ernst, R. Chollet, Substrate specificity and regulation of the maize (Zea mays) leaf ADP:protein phosphotransferase catalyzing phosphorylation/inactivation of pyruvate,orthophosphate dikinase, Biochem. J. 236 (1986) 579–584.