Acetyl CoA provides maximum activation at about 1 mM concentration, but we were unable to detect significant effects with the fructose diphosphate, guanosine diphosphate, or even the aspartate.
Dioxane is very effective, and is used in the assay. However, dioxane generally contains traces of peroxides which must be removed by the dithioerythritol DTE. See also Wohl and Markus Specificity : Apparently only phosphoenolpyruvate and carbon dioxide can participate in the reaction. Place Order. Phosphoenolpyruvate Carboxylase I. Joonho Park ; Joonho Park. William C. Plaxton William C. Plaxton 1. Biochem J 1 : 15— Article history Received:.
Revision Received:. Get Permissions. You do not currently have access to this content. View full article. Sign in Don't already have an account? Sign in to your personal account. You could not be signed in. Sign In Reset password. Sign in via your Institution Sign in via your Institution. Taken together with the phylogenetic analysis, this change in transcription supports the conclusion that PPCKA is the isoform that was recruited in the transition from C 3 to C 4 photosynthesis mode.
In addition to the maximal transcript accumulation at night in both species, a decrease in transcript levels is observed from the C 3 to the C 4 species Fig. Thus, it appears that this isoform has decreased importance in the leaves of the newly evolved C 4 Flaveria spp. On the other hand, PPCKB is fully functional in the C 3 species, where nighttime phosphorylation is relatively abundant.
As a side finding, the existence of daytime- and nighttime-expressed PPCK in the C 3 species, especially if this expression pattern for the PPCK family is confirmed for other C 3 plants, is an explanation for the varying PEPC phosphorylation results observed among various C 3 plants Fukayama et al.
An analysis of cis-regulatory elements in the PPCK promoter regions would also be useful to investigate any regulatory differences between Flaveria spp. Thus, in both cases, we found indications for rapid transcript turnover that agree with a previously postulated theory Tsuchida et al. Eight of the 13 amino acid exchanges conserved among the PPCKA proteins from C 3 and C 4 plants are concentrated in the region of amino acids to Fig.
Some of the potentially important differences are at positions , to , and At position there is a Ser-to-Gly exchange. In contrast to the nonpolar amino acid Gly, Ser is a polar amino acid that can be subject to phosphorylation. Moreover, intramolecular or intermolecular hydrogen bonds can be formed with the Ser side chain. At positions , , and , three amino acid exchanges were identified.
The first two are exchanges among amino acids with similar chemical properties, but the last exchange, at position , replaces Gly, a small nonpolar amino acid, with Arg, a larger basic polar amino acid, whose side chain is fully protonated at neutral pH, consequently bringing in positive charge at this position Taniguchi, This cluster of point mutations is followed by an Arg-to-Met exchange at position In the three-dimensional structure, Arg is found opposite the region to Together with a third amino acid exchange at position , these amino acid exchanges are predicted to lie in an unstructured protein region, partly annotated as the activation loop A-loop ; Fig.
Current structural annotations state that phosphorylation of the A-loop results in transition from a disordered to an ordered state of the kinase and is part of the kinase activation mechanism accession no. As the putative A-loop contains Ser positions and , Thr positions and , and Tyr position , which are all susceptible to phosphorylation, the nearby exchanges might influence the interaction with another regulating protein.
The latter will be necessary for proper determination of the influence of these amino acid exchanges in the tertiary structure of PPCK. If we assume that the evolutionary trajectories are accompanied by gains of fitness and that the evolution of the PPCK gene family optimized the C 4 evolutionary processes Sage et al.
Starting from a common ancestor in the genus Flaveria , species with C 3 photosynthesis diverged earlier, whereas the C 4 species diverged later. If an amino acid exchange is conserved among all intermediates and the C 4 species type 1 , then this point mutation probably occurred before those found only in the C 4 species type 3; Table I.
In the two cases where the C 4 residue is found in some but not all intermediate species ED and SN; type 2 , it is difficult to determine what happened. Creating a series of mutated PPCKA proteins will be needed to investigate the importance of each of these amino acid exchanges for the kinase enzymatic activity.
The recombinant PPCKA experiments presented here show that these amino acid differences are not enough to abolish substrate recognition among the species in the genus Flaveria Fig. Since a higher signal results from the C 4 form of both kinase and substrate, it is the parallel optimization of these molecules that is responsible for this increased activity Fig.
In this context, the so-far ignored amino acid differences in the N terminus of PEPC, and in the vicinity of the phosphorylated Ser Fig.
Simulation of the C 3 -to-C 4 exchanges on the N-terminal part of PEPC that take into account that PEPC is a tetramer show that, despite the macromolecular conformation, these amino acids would still be accessible for protein-protein interaction Fig. Supplemental File S7. The side chains of these two amino acids have similar properties, but the smaller side chain from Val might allow easier uncoupling of the kinase from the substrate, by providing larger separation between the PEPC- and PPCK-interacting residues.
Consequently, this might be one of the reasons for observing a stronger signal in the in vitro phosphorylation assay between the C 4 recombinant PEPC and PPCK proteins. This introduces a positive charge close to Ser, but the true consequence of this exchange will only be known after further investigation, as it will depend on the interacting residue from PPCK.
Cocrystallization of both proteins should uncover the amino acid residues involved in the protein-protein interaction and allow the investigation of the observed amino acid exchanges between isoforms from C 3 and C 4 plants. Additionally, as the cumulative findings from previous and these studies reinforce the differences in phosphorylation patterns of PEPC from C 3 and C 4 species, and at a time with ongoing efforts for engineering C 4 traits in C 3 crops, we can only stress the need for detailed knowledge of the regulation of one of the key enzymes of the C 4 pathway.
Leaf blades were harvested from wild-type 4- to 6-week-old plants from Flaveria trinervia , Flaveria ramosissima , and Flaveria pringlei. Leaf samples were collected at 4-h intervals over a h period. Three separate biological replicates for each time point were ground in liquid nitrogen prior to the respective RNA and protein preparations. Total RNA was extracted from Flaveria spp. Digestion with DNase was performed for 15 min. RS, part no. A, November RNA aliquots and libraries were validated for qualification and quantitation with the Agilent bioanalyzer Agilent Technologies.
Validated DNA libraries were normalized and pooled to a final concentration of 2 n m. Single-read sequencing was performed with the Illumina HiSeq These were then combined and aligned to the F. AB , allowing us to combine the contigs of the two pipelines for several species and ensure the largest possible coverage of the PPCK transcript. Mallmann, D. Heckmann, A. Lercher, A. Weber, P. Westhoff, and U.
Gowik, unpublished data. To obtain the abundance of reads for the kinase, clean Illumina reads were aligned against F. Reads were mapped with Bowtie2 version 2. We counted the number of unambiguous best hits per transcript and normalized these values to reads per million mapped reads and kilobase transcript length RPMK. Means and se were calculated from three biological replicates.
Total RNA was extracted from F. All generated plasmids were verified by DNA sequencing. The QIAexpressionist protocol was used for the heterologous expression of native protein Qiagen. In short, intact E. The cells were disrupted by sonification. In order to protect protein integrity, a gradient from 0 to m m imidazole in 50 m m steps was used.
Signals were quantified using the program Multi Gauge 3. Immunoblot analysis was examined using the reagent of the chemiluminescence assay SuperSignal West; Thermo Scientific. Unfortunately, although specific, the antibody sensitivity was determined to be above 0. Initial testing was performed on the recombinant proteins for F.
The peptides that showed good properties in the mass spectrometer were checked against the Flaveria spp. The peptides then underwent a second round of selection using plant extract, and those peptides that could be successfully monitored in this complex sample were obtained as heavy peptides 13 C and 15 N from Thermo Fisher Scientific. None of the peptides from PPCK could be monitored in complex plant extracts using our setup.
Proteins were extracted from F. Prior to desalting, the synthetic peptides for PEPC listed in Supplemental File S6 were added to the samples in species-specific amounts.
Per ng of total protein, the amounts of synthetic peptides were as follows: F. Samples were sprayed into the mass spectrometer by electrospray ionization. The resolution of Q1 and Q3 was set to 0. Cycle time was 1 s dwell time was more than 50 ms for each transition. Peptides were fragmented using argon gas for collision-induced dissociation at collision energies specific for each peptide Supplemental File S6. Instrument scan mode was iSRM, and three primary and two secondary fragment ions per peptide were collected; allowed time was 0.
Data were analyzed using PinPoint 1. The following materials are available in the online version of this article. Supplemental File S1. Supplemental File S2. Phylogenetic analysis of plant PPCKs. Supplemental File S3. Alignment of PPCKs from various species. Supplemental File S4. Supplemental File S5.
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