Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity (original) (raw)
Related papers
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
Nucleic Acids Research, 1986
A recombinant clone, pM52, containing cDNA for maize phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) was isolated f rom a maize leaf cDNA library constructed using an expression vector in Escherichia coli. The screening of the clone was conveniently performed through its ability to complement the phenotype (glutamate requirement) of PEPCase-negative mutant of E. coli. The enzyme encoded by this clone was identical with the major PEPCase in maize, a key enzyme in the C4-pathway, as judged from its allosteric properties and immunological reactivity. The cloned cDNA (3093 nucleotides in length) contained an open reading frame of 2805 nucleotides, the 3'-untranslated region of 222 nucleotides and the poly(dA) tract of 64 nucleotides. The deduced amino acid sequence (935 residues) of the enzyme showed higher homology with that of an enterobacterium, E. coli (43%) than that of a cyanobacterium (blue-green alga), Anacystis nidulans (33%).
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...
European Journal of Biochemistry, 1995
Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8→Asp) forms of purified recombinant sorghum C 4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.3 1) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent K i for the negative allosteric effector l-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C 4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and K m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by l-malate and, most especially, K a (Glc6P) and K i (l-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C 4 -photosynthesis enzyme.
PLANT PHYSIOLOGY, 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
Photosynthesis research, 2003
Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C(4)-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize PEPC was functioning even though it remained dephosphorylated and less active in the light. (14)CO(2) labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO(2) fixation of transgenic rice plants. Rather, it slightly lowered the CO(2) assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marke...
Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms
Archives of Biochemistry and Biophysics, 2003
Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate and Pi using Mg 2þ or Mn 2þ as a cofactor. PEPC plays a key role in photosynthesis by C4 and Crassulacean acid metabolism plants, in addition to its many anaplerotic functions. Recently, three-dimensional structures of PEPC from Escherichia coli and the C4 plant maize (Zea mays) were elucidated by X-ray crystallographic analysis. These structures reveal an overall square arrangement of the four identical subunits, making up a ''dimer-of-dimers'' and an eight-stranded b barrel structure. At the Cterminal region of the b barrel, the Mn 2þ and a PEP analog interact with catalytically essential residues, confirmed by site-directed mutagenesis studies. At about 20 A A from the b barrel, an allosteric inhibitor (aspartate) was found to be tightly bound to downregulate the activity of the E. coli enzyme. In the case of maize C4-PEPC, the putative binding site for an allosteric activator (glucose 6-phosphate) was also revealed. Detailed comparison of the various structures of E. coli PEPC in its inactive state with maize PEPC in its active state shows that the relative orientations of the two subunits in the basal ''dimer'' are different, implicating an allosteric transition. Dynamic movements were observed for several loops due to the binding of either an allosteric inhibitor, a metal cofactor, a PEP analog, or a sulfate anion, indicating the functional significance of these mobile loops in catalysis and regulation. Information derived from these three-dimensional structures, combined with related biochemical studies, has established models for the reaction mechanism and allosteric regulation of this important C-fixing enzyme.
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.
FEBS Letters, 1997
In order to mimic regulatory phosphorylation of the Ser-15 of maize C 4-form phosphoewo/pyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wildtype PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition.