Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize (original) (raw)
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Structure, 2002
itive reaction of the carboxylation through the oxygenation of ribulose 1,5-bisphosphate carboxylase/oxy-Osaka University Suita, Osaka 565-0871 genase (Rubisco). The photorespiration decreases the efficiency of CO 2 assimilation and is increased under Japan 2 Department of Public Health environmental conditions such as water stress and high temperature [4]. In contrast, C 4 plants have a unique Graduate School of Medicine Kyoto University atmospheric CO 2 pathway involved in PEPC. Unlike Rubisco, PEPC uses HCO 3 Ϫ , instead of CO 2 , as a sub-Sakyo-ku, Kyoto 606-8501 Japan strate and has a high affinity for the relatively inert bicarbonate ion. C 4 form PEPC is more abundantly expressed 3 Division of Integrated Life Science Graduate School of Biostudies in C 4 plants than in C 3 plants and catalyzes the first committed step for the fixation of atmospheric CO 2 dur-Kyoto University Sakyo-ku, Kyoto 606-8502 ing C 4 photosynthesis [2]. In C 4 plants, after carboxylation by PEPC, the resulting C 4 compounds are trans-Japan ferred into the chloroplast in bandle sheath cells and decarboxylated to supply Rubisco with a high concentration of CO 2 [5]. As a consequence, Rubisco oxygen-Summary ation is more effectively suppressed in C 4 plants. The reaction mechanism of PEPC for carboxylation has re-Phosphoenolpyruvate carboxylase (PEPC) catalyzes ceived much attention because PEPC has a high affinity the first step in the fixation of atmospheric CO 2 during for HCO 3 Ϫ and is not inhibited by O 2. C 4 photosynthesis. The crystal structure of C 4 form Recently, trials for introducing C 4-specific genes into maize PEPC (ZmPEPC), the first structure of the plant C 3 plants have been carried out to improve the efficiency PEPCs, has been determined at 3.0 Å resolution. The of CO 2 fixation in C 3 photosynthesis through recombistructure includes a sulfate ion at the plausible binding nant DNA techniques [6]. PEPC genes were also overexsite of an allosteric activator, glucose 6-phosphate. pressed in C 3 plants, such as rice, potato, and tobacco The crystal structure of E. coli PEPC (EcPEPC) com-[7-10]. In particular, high-level expression of maize plexed with Mn 2؉ , phosphoenolpyruvate analog (3,3-PEPC transgenic rice plants was reported to show a dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), reduction of O 2 inhibition of photosynthesis [7]. In transand an allosteric inhibitor, aspartate, has also been genic potatos, overexpression of the PEPC gene redetermined at 2.35 Å resolution. Dynamic movements sulted in the induction of other C 4 enzymes [10]. Since were found in the ZmPEPC structure, compared with the activities of PEPC in transgenic plants are also reguthe EcPEPC structure, around two loops near the aclated by metabolites, such as malate and glucose tive site. On the basis of these molecular structures, 6-phosphate, and covalent modification by reversible the mechanisms for the carboxylation reaction and for phosphorylation, as in C 4 plants, the properties of PEPC the allosteric regulation of PEPC are proposed. need to be modified by gene engineering suitable for the transgenic plants. Thus, an understanding of the Introduction molecular mechanism of PEPC has been crucial in elucidating the regulation mechanism. PEPC (EC 4.1.1.31) catalyzes the irreversible HCO 3 Ϫ-All known PEPCs are tetrameric enzymes with molecdependent/biotin-independent carboxylation of phosular weights of 000,044ف [3]. The amino acid sequences phoenolpyruvate (PEP) in the presence of a divalent of these PEPCs show significant conservation [11]. For cation, such as Mg 2ϩ or Mn 2ϩ , to form oxaloacetate example, ZmPEPC and EcPEPC share 40% identity (Fig-(OAA) and phosphate (Figure 1A) [1-3]. The enzymes ure 2), suggesting that the reaction mechanisms among have been isolated from various organisms, including the enzymes from various organisms are essentially the plants and a variety of bacteria. PEPC in nonphotosynsame. In most cases, the activity of the enzyme is allothetic tissues takes on anaplerotic functions by replensterically controlled by a variety of positive (e.g., glucose ishing C 4 dicarboxylic acids for the syntheses of various 6-phosphate) and negative (e.g., malate and aspartate) cellular constituents and for the maintenance of the citric metabolite effectors. Despite the knowledge of the crysacid cycle [1-3]. On the other hand, higher plants have tal structure of the EcPEPC complexed with aspartate several isoforms of PEPC with different kinetic and regulatory properties that correlate with their respective roles
Plant physiology, 2003
Phosphoenolpyruvate carboxylase (PEPC) is distributed in plants and bacteria but is not found in fungi and animal cells. Important motifs for enzyme activity and structure are conserved in plant and bacterial PEPCs, with the exception of a phosphorylation domain present at the N terminus of all plant PEPCs reported so far, which is absent in the bacterial enzymes. Here, we describe a gene from Arabidopsis, stated as Atppc4, encoding a PEPC, which shows more similarity to Escherichia coli than to plant PEPCs. Interestingly, this enzyme lacks the phosphorylation domain, hence indicating that it is a bacterial-type PEPC. Three additional PEPC genes are present in Arabidopsis, stated as Atppc1, Atppc2, and Atppc3, encoding typical plant-type enzymes. As most plant PEPC genes, Atppc1, Atppc2, and Atppc3 are formed by 10 exons interrupted by nine introns. In contrast, Atppc4 gene has an unusual structure formed by 20 exons. A bacterial-type PEPC gene was also identified in rice (Oryza sat...
Plant and Cell Physiology, 1998
A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase (PEPC) was isolated. In the coding region, the root-form PEPC showed 76 and 77% identity with the C 4-and C 3-form PEPCs of maize, respectively, at the nucleotide level. At the amino acid level, the root-form was 81 and 85% identical to the C 4-and C 3-form PEPCs, respectively. The entire coding region was inserted into a pET32a expression vector so that it was expressed under the control of T7 promoter. The purified recombinant root-form PEPC had a V max value of about 28 //mol min" 1 (mg protein)" 1 at pH 8.0. The K m values of root-form PEPC for PEP and Mg 2+ were one-tenth or less of those of C-form PEPC when assayed at either pH 7.3 or 8.0, while the value for HCOf was about one-half of that of C 4form PEPC at pH 8.0. Glucose 6-phosphate and glycine had little effect on the root-form PEPC at pH 7.3; they caused twofold activation of the C 4-form PEPC. The K, (L-malate) values at pH 7.3 were 0.12 and 0.43 raM for the root-and C 4-form PEPCs, respectively. Comparison of hydropathy profiles among the maize PEPC isoforms suggested that several stretches of amino acid sequences may contribute in some way to their characteristic kinetic properties. The root-form PEPC was phosphorylated by both mammalian cAMP-dependent protein kinase and maize leaf protein kinase, and the phosphorylated enzyme was less sensitive to L-malate.
Biochimie, 2010
Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO3, OAA and ITP with a K m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.
The Plant Journal, 2000
The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C 4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-speci®c antibodies to the C 4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that¯anking the speci®c site of regulatory phosphorylation (Ser15) and subsequently puri®ed by af®nity-chromatography. Speci®city of the resulting antibodies to the C 4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the ®eld, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C 4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.
Journal of Experimental Botany, 2007
Introducing a C 4-like pathway into C 3 plants is one of the proposed strategies for the enhancement of photosynthetic productivity. For this purpose it is necessary to provide each component enzyme that exerts strong activity in the targeted C 3 plants. Here, a maize C 4-form phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was engineered for its regulatory and catalytic properties so as to be functional in the cells of C 3 plants. Firstly, amino acid residues Lys-835 and Arg-894 of maize PEPC, which correspond to Lys-773 and Arg-832 of Escherichia coli PEPC, respectively, were replaced by Gly, since they had been shown to be involved in the binding of allosteric inhibitors, malate or aspartate, by our X-ray crystallographic analysis of E. coli PEPC.
Plant Science, 2002
A phosphoenolpyruvate carboxylase gene (referred to as Ppc1) was isolated from a wheat genomic library and sequenced in both strands. The deduced protein sequence is encoded by 10 exons and shows extensive similarities to that of other plant PEPCs sequenced so far, with respect to the predicted motives involved in the structure (b-strands and a-helices) and function (active site, inhibitor site, N-and C-terminus) of the enzyme. Modelling of the 3D-structure of the wheat PEPC monomer using the Swiss-Model programme and the Escherichia coli PEPC monomer 3D-structure as a reference revealed that these motives and corresponding key residues share very similar positions in the folded plant and bacterial polypeptides. Interestingly, the 5%-flanking sequence of Ppc1 gene contains various putative regulatory cis-elements including an ABRE box. Relative RT-PCR analysis showed a ubiquitous expression of Ppc1 gene in developing and germinating wheat seeds as well as in developing seedlings.
1991
Reversible seryl-phosphorylation contributes to the light/dark regulation of C4-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malatesensitivity of the target enzyme has been recently identified as Ser-15 in 32P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [32P]phosphopeptides were isolated and purified from in vivo 32P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more 32P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residu...
The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize
Photosynthesis Research, 2006
The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K0.5 PEP for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K0.5 PEP, while the K0.5 PEP of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme’s kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.
Phosphoenolpyruvate carboxylase: structure, regulation and evolution
Plant Science, 1994
Plant phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is encoded by a small multigene family in which the expression of each member is controlled individually by exogenous (light, environmental) and/or endogenous (hormonal and developmental) stimuli. The involvement of putative trans-acting factors and consensus cis-elements of promoters in the specific transcriptional regulation of the PEPC genes is discussed. At the post-translational level, the regulatory strategy of the plant enzyme is mainly to offset the negative effect of the feedback inhibitor, L-malate, the end-product of the oxaloacetate reduction. All plant PEPC-forms are under positive and negative allosteric control by metabolite effectors and possess a consensus phosphorylation site containing a target serine residue near their Nterminus (e.g. Ser8 in C4 PEPC from sorghum). In C 4 and Crassulacean acid metabolism (CAM) plants, a complex signal-transduction chain activates ~a Ca2+-independent protein-serine kinase responsible for regulatory phosphorylation of PEPC. A more thorough understanding of the functional and regulatory properties of the bacterial and C 4 enzymes has emerged by exploiting recombinant proteins and site-directed mutagenesis. In these newly opened areas, PEPC offers one of the best characterized paradigms of plant signaling. Finally, some emerging ideas on the evolution and phylogenetic relationships of the various PEPC isoforms are presented.