JXB Advance Access originally published online on April 11, 2008
Journal of Experimental Botany 2008 59(7):1811-1818; doi:10.1093/jxb/ern018
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity
1Laboratory of Plant Physiology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
2Laboratory of Plant Physiology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
3Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8526, Japan
4Department of Materials Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
5Department of Biotechnological Science, Kinki University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan
To whom correspondence should be addressed. E-mail: izui{at}waka.kindai.ac.jp
Received 3 December 2007; Revised 6 January 2008 Accepted 11 January 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Introducing a C4-like pathway into C3 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 C3 plants. Here, a maize C4-form phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.3 [EC] 1) was engineered for its regulatory and catalytic properties so as to be functional in the cells of C3 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. The resulting mutant enzymes were active but their sensitivities to the inhibitors were greatly diminished. Secondly, a Ser residue (S780) characteristically conserved in all C4-form PEPC was replaced by Ala conserved in C3- and root-form PEPCs to decrease the half-maximal concentration (S0.5) of PEP. The double mutant enzyme (S780A/K835G) showed diminished sensitivity to malate and decreased S0.5(PEP) with equal maximal catalytic activity (Vm) to the wild-type PEPC, which will be quite useful as a component of the C4-like pathway to be introduced into C3 plants.
Key words: C4 photosynthesis, genetic engineering, PEP carboxylase, site-directed mutagenesis, Zea mays
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.3 [EC] 1) catalyses an irreversible β-carboxylation of phosphoenolpyruvate (PEP) in the presence of HCO
and Mg2+ to yield oxaloacetate and orthophosphate (O'Leary, 1982). In higher plants, the enzyme has many faceted physiological roles, which are shared by specific isoforms. Particularly in C4- and crassulacean acid metabolism (CAM) plants, PEPC plays a key role in photosynthesis by performing the initial fixation of atmospheric CO2. Most PEPCs are subject to allosteric regulation. Their effectors show a wide variety depending on the species of organisms (Izui et al., 2004), and they often affect kinetic properties differently, even among isoforms in the same organism (Dong et al., 1998; Svensson et al., 2003). However, the effectors of higher plant PEPCs are confined to a set of metabolites; namely, PEPCs of dicot plants are activated by glucose 6-phosphate (G6P) and inhibited by malate or aspartate, and those of monocot plants are further activated by glycine. Furthermore, higher plant PEPCs are regulated by reversible phosphorylation by a specific protein kinase called PEPC kinase (PEPC-k), and the phosphorylated PEPC shows lower sensitivity to malate and aspartate (Vidal and Chollet, 1997; Tsuchida et al., 2001; Nimmo, 2003). Recently, PEPC-k knockdown Flaveria bidentis transgenic plants were produced using reverse genetics, and no obvious differences were observed in several photosynthetic parameters between transgenic plants and non-transformants, at least under normal greenhouse conditions (Furumoto et al., 2007). For the coming global shortage of food in the near future, a second Green Revolution is required, which may bring about a tremendous improvement in the productivity of crop plants. The photosynthetic productivity of C4 plants is usually 1.5–2-fold higher than that of C3 plants under arid, hot, and light-intensive conditions owing to their CO2-concentrating metabolic cycle, the C4 pathway. For this reason, several groups of investigators have been trying to use genetic engineering to introduce a ‘C4-like pathway’ into C3 plants that have no Kranz anatomy (Surridge, 2002). The ‘C4-like pathway’ is supposed to function in a single mesophyll cell and ultimately pumps p CO2 from the cytoplasm to the chloroplasts where the net CO2-fixation is performed. The discovery of this pathway in Hydrilla verticillata by Bowes et al. (2002) reinforced the feasibility of this strategy. Theoretical analysis by von Caemmerer (2003) also supported some advantages of this approach under water-limited conditions.
Previously, tobacco transformants expressing maize C4-form PEPC were first produced, but no enhancement of photosynthetic activity could be seen (Kogami et al., 1994). Although several genes for the enzymes involved in the C4 pathway had been successfully introduced and expressed in C3 plants individually or in combination, thereafter, no substantial improvement in photosynthesis due to an operational ‘C4-like pathway’ has been obtained (reviewed by Matsuoka et al., 2001; Häusler et al., 2002; Miyao and Fukayama, 2003; Raines, 2006). During the course of these experiments, several problems to be solved became apparent. One is that the overexpressed foreign enzymes do not necessarily exert their expected catalytic activities in the targeted C3 cells, since they were not well matched with cellular levels of ligands. Thus it is necessary to construct engineered enzymes of the ‘C4-like pathway’, whose catalytic and regulatory properties are modified to be able to function in any given intracellular environment of C3 plants (Häusler et al., 2002). In the case of PEPC, there was a very small effect of overexpression of maize C4-form PEPC on the cellular metabolism of rice and this was presumed to be because the intracellular levels of PEP and malate were too low and too high, respectively, for the functioning of the maize C4-form PEPC (Fukayama et al., 2003). In fact, the Km value of this enzyme for PEP is 1.5 mM, being about 30-fold larger than that of a non-photosynthetic form (root-form) PEPC of maize, and the Ki value for malate is 0.8 mM, being about 4-fold larger than the root-form PEPC (Dong et al., 1998). The same tendency was also observed with several isoform PEPCs of Flaveria trinervia, a C4 dicot plant (Bläsing et al., 2000).
In addition, all of the transformants analysed to date show high levels of malate, which is enough to diminish PEPC activity in vitro (Kogami et al., 1994). In the intracellular environment, the foreign C4-form PEPC appears not to be fully active. Therefore, it is worthwhile to prepare an engineered enzyme, which fits to the ‘C3 environment’ and especially the low PEP- and high malate concentrations, for further metabolic engineering.
For molecular engineering on the enzyme, it is important to determine the key residues that influence these catalytic and regulatory properties. In recent years, a Ser residue involved in affinity for PEP was identified in the F. trinervia C4-form PEPC (at position 774, corresponding to 780 in the maize C4-form PEPC) (Bläsing et al., 2000). This Ser residue is conserved characteristically in C4-isoform enzymes among various plant species, while in other isoforms this site is an Ala residue. Replacement of this Ser of C4-isoform PEPC to Ala successfully decreased its Km value for PEP (Bläsing et al., 2000).
Aspartate and malate are common allosteric inhibitors in both maize and Escherichia coli enzymes. The aspartate-binding site was first revealed from the analysis of the crystal structure of the E. coli enzyme (Kai et al., 2003). Both the crystal structures of E. coli PEPC (Kai et al., 1999) and the maize C4-form PEPC (Matsumura et al., 2002) were determined. The former was resolved as an inactive state complexed with aspartate, while the latter was resolved as an active state not complexed with aspartate. In the E. coli PEPC structure, aspartate was ligated with four residues, Arg-587, Lys-773, Arg-832, and Asn-881. These residues are widely conserved among PEPCs. Two of them, Arg-587 and Asn-881 are indicated to be essential for catalytic activity as well. The replacement Arg-587 to Ser diminished its catalytic activity (Yano et al., 1995). In the case of a sorghum C4-isoform enzyme, deletion of the four C-terminal residues, which included an Asn corresponding to Asn-881 of maize C4-form PEPC, destroyed its catalytic activity (Dong et al., 1999). As for the residues, Lys-773 and Arg-832, there has been no experimental evidence to show their involvement in the aspartate binding, except for our structural analysis.
In this report, mutant E. coli PEPCs were first produced with either Lys-773 or Arg-832 replaced by Gly. Then mutant maize PEPCs were produced with corresponding residues, Lys-835 and Arg-894, replaced in the same way. The maize PEPC showed greatly diminished inhibitor sensitivity as a result of replacement of these residues.
Based on these findings, the intention was to produce an engineered maize C4-form PEPC with altered affinity to ligands, which would exert its activity sufficiently in the C3 environment of host plants. To this end, two residues of the maize C4-form PEPC were substituted independently or simultaneously. Lys-835 which is involved in Asp binding was substituted with Gly; and Ser-780 which is involved in PEP affinity was substituted with Ala. The resulting recombinant enzymes were investigated for their kinetic properties. The double mutant enzyme showed the higher affinity for PEP and lower sensitivity to allosteric inhibitors than the wild-type enzyme (WT), demonstrating for the first time that these properties could be conferred to the enzyme almost additively. The possible use of this mutant enzyme in metabolic engineering is discussed.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Construction of plasmids
The enzymes, chemicals, and bacterial strains in this study were used according to the previous report (Takahashi-Terada et al., 2005). The following pT3, pTM94 and pEM94 (Dong et al., 1997) derivatives were constructed by site-directed mutagenesis in this work; pT3-K773G, pT3-R832G, pEM-K835G, pEM-R894G, pEM-K835G/R894G, pEM-S780A, and pEM-S780A/K835G. The original pT3 plasmid contains a major part of the gene (ppc) for E. coli PEPC, consisting of the coding region and a part of the promoter region in pUC18 (Takara) (Terada et al., 1995). The original pEM94 plasmid contains the complete coding region of the maize C4-form PEPC in pET32a (Novagen) (Dong et al., 1997). The site-directed mutagenesis was carried out by the overlap extension method using PCR (Ho et al., 1989). The mutagenized primer sets used in this work were as follows: 5'-TTCGCCggAGCAGACCTGTGG-3' and 5'-CAGGTCTGCTccGGCGAAGACC-3' for pT3-K773G, 5'-ATTCAGCTAgGGAATATTTAC-3' and 5'-AAATATTCCcTAGCTGAATAGAC-3' for pT3-R832G, 5'-GAACCTGGTCTGGGTCCACGcGAAGATCCA-3' and 5'-CTGGATCTTCgCGTGGACCCAGACC-3' for pEM-K835G, 5'-GGCTGGTGCTGgGCAACCCCTACATCAC-3' and 5'-GTGGTGATGTAGGGGTTGCcCAGCACC-3' for pEM-R894G, 5'-GAACCTGGTCTGGGTCCACGcGAAGATCCA-3' and 5'-CTGGATCTTCgCGTGGACCCAGACC-3' for pEM-S780A. The mutated bases are shown by lowercase letters. The upstream primer and the downstream primer sets in the second PCR were as follows: 5'-TCAATCCGCGCCATTCCGTGG-3' and 5'-TCTTCTTCTTGCAGGTTGCG-3' for pT3-K773G, 5'-GGAGATGGTCTTCGCCAAAGCAGACC-3' and 5'-GATTAGCCGGTATTACGCATACC-3' for pT3R832G, 5'-CACGTTCGGGCTCTCCCTGGTGAAG-3' and 5'-CGAAAACCATCTCCAGCAGG-3' for pEM-K835G, 5'-CGACGGCCAGTGAATTCG-3' and 5'-GTTCGCCATCGACAAGGACGT-3' for pEM-R894G, 5'-CGAAAACCATCTCCAGCAGG-3' and 5'-CACGTTCGGGCTCTCCCTGGTGAA-3' for pEM-S780A, respectively. In the case of preparing pEM-K780A, a primer set of 5'-GATCGCCGACGTCATCGCCGCGTTCCAC-3' and 5'-CACGTTCGGGCTCTCCCTGGTGAA-3' was used for the second PCR to reduce artificial PCR products. After cloning of the amplified mutagenized fragments into the pGEM-T Eazy Vector System (Promega), the sequences of the fragments were confirmed. The cloned fragments were then excised by digestion with SphI and BamHI (for pT3-K773G and pT3-R832G), with NcoI and HindIII (for pEM-R894G, pEM-K835G, and pEM-R894G), or with AatII (for pEM-S780A), and then inserted into corresponding sites of pEM94 or pTM94. The latter plasmid was used for expression of the maize C4-form PEPC in E. coli F15 (
ppc), a deletion mutant of the ppc gene (Terada et al., 1995).
Complementation test of mutant PEPCs for growth of E. coli F15 (ppc), expression and purification of recombinant PEPCs
Procedures for the complementation test of mutant PEPCs, their expression and their purification were the same as described previously by Takahashi-Terada et al. (2005). The genes for WT and mutant PEPCs of E. coli and maize were cloned into the pT3 and pTM plasmids, respectively, and expressed in E. coli F15. Since the E. coli PEPC protein comprises about 30% of the total soluble protein, the crude cell extracts were used for assays without further purification. On the other hand, WT and mutant maize enzymes were expressed in E. coli (BL21DE3) with the pET system (Novagen). These enzymes were purified with a Ni2+-chelating affinity column (GE Healthcare, UK) as a fusion protein with tags of 159 amino acid residues at the N-terminus. Our previous studies had shown that the kinetic properties are not significantly affected by this artificial tag peptide (Dong et al., 1997). Therefore, the purified enzymes were used for kinetic analysis without truncation of the tag peptide. The purity of each recombinant enzyme was estimated to be more than 90% by SDS–PAGE (data not shown). Although the double mutatnt maize enzyme was not accumulated in E. coli F15, this protein could be successfully accumulated in the other host E. coli BL21 used in the pET system, and purified in the same manner as the other mutant enzymes. The molecular size of the purified protein coincided with the expected size of 127 kDa (with a 17 kDa artificial tag) on an SDS-PAGE (data not shown). Each litre of bacterial culture yielded approximately 2 mg of up to 90% pure protein.
Measurement of PEPC activity
PEPC activity was measured spectrophotometrically as described by Terada and Izui (1991) and Takahashi-Terada et al. (2005). Briefly, the standard assay mixture for E. coli PEPC contained, in a total volume of 1.0 ml, 100 mM TRIS–HCl (pH 8.5), 2 mM PEP, 1.0 mM acetyl-CoA (an allosteric activator), 10 mM KHCO3, 10 mM MgSO4, 0.1 mM NADH, 1.5 IU of malate dehydrogenase, and the enzyme. The assay mixture for maize PEPC contained, in a total volume of 1.0 ml, 100 mM HEPES-NaOH (pH 7.3), 2 mM PEP, 10 mM KHCO3, 10 mM MgSO4, 0.1 mM NADH, 1.5 IU of malate dehydrogenase, and the enzyme. The reaction was initiated by the addition of 2 mM or 0.5 mM PEP. The protein concentration was routinely determined using a protein assay kit (Bio-Rad) based on dye binding (Bradford, 1976) with crystalline bovine serum albumin as a standard. When necessary, the molar concentration of the maize C4-form PEPC subunit was estimated from its molar extinction coefficient at 280 nm (7.5x104 M–1 cm–1). The value had been determined previously by amino acid analysis of the acid hydrolysate of PEPC in a solution of known absorbance (Y Ueno, K Izui, unpublished data). More simply, the PEPC concentration of 1.0 mg ml–1 estimated by the dye-binding method above corresponds to an absolute concentration of 0.72 mg ml–1, and this solution gives an absorbance of 0.5 at 280 nm.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Complementation test of mutant enzymes
Mutant PEPCs of E. coli, K773G and R832G, as well as wild-type PEPC (WT) could complement the glutamate-requiring phenotype of E. coli F15, a deletion mutant of the gene for PEPC (Fig. 1A). This indicates that the mutant enzymes were active, at least to the extent to support cell growth. Among the maize mutant PEPCs, K835G complemented well and R894G complemented slightly, whereas the double mutant K835G/R894G did not (Fig. 1B). Crude protein extract was prepared from each of the cell lines grown on the agar plate containing glutamate (Fig. 1B), and subjected to the western blot analysis using the anti-maize PEPC antibody (Fig. 1C). The signals for PEPCs, WT, K835G, and R894G, were detected with an expected protein size, while that of the double mutant was not. Thus R894G was accumulated in the E. coli cells, but the enzyme activity was insufficient to support good growth of the mutant F15. On the other hand, no accumulation of the double mutant K835G/R894G was observed, the reason for which remains unknown.
|
Kinetic properties of enzymes with mutations at the inhibitor binding site
The crude extracts from the transformed F15 cells which expressed E. coli mutant PEPCs, either K773G or R832G, were assayed for PEPC activity, since each of the mutant PEPCs was able to complement the F15 phenotype. However, only a very low activity was observed for K773G probably due to its instability in the cell extracts. For R832G, a high activity was observed. The concentrations required for 50% inhibition (I0.5) for aspartate and malate in the presence of 1 mM acetyl-CoA were 1.7 mM and 1.8 mM, respectively, for the WT PEPC, while those values increased to 15.3 mM and 3.6 mM for R832G. In the absence of acetyl-CoA, the extent of the desensitization became more profound for both inhibitors (data not shown).
Maize mutant PEPCs, whose putative residues involved in the binding with allosteric inhibitor (Lys-835 and Arg-894) had been replaced with Gly, were purified to homogeneity and their sensitivity to malate or aspartate was investigated. As shown in Fig. 2 and Table 1, remarkable desensitization to these inhibitors was observed. Further kinetic measurements showed that their major catalytic properties were retained even after these mutations.
|
|
Effect of replacement of Ser-780 to Ala on kinetic properties
For the C4-form PEPC from a dicot plant F. trinervia, replacement of the conserved Ser, which corresponds to Ser-780 of the maize PEPC, to Ala had been shown to decrease the concentration of PEP required for half-maximum velocity (S0.5) (Bläsing et al., 2000). To test whether this also holds true for PEPC from a monocot plant, maize, a mutant enzyme S780A was produced and its kinetic properties were investigated. As shown in Fig. 3A and Table 2, the S0.5 value of S780A was decreased to about one-quarter of WT. The saturation curve of S780A for Mg2+ was weakly sigmoidal, similarly to WT but the S0.5 value was not changed (Fig. 3B). The apparent increase of Vmax for S780A relative to WT was due to the difference in the degree of PEP saturation, since the PEP concentration employed was sufficiently low so that the extents of its saturation were different from each other. The I0.5 values for S780A were also apparently slightly larger than WT for the same reason. It is known that the I0.5 values increase with increasing concentrations of PEP through heterotrophic interaction between these ligands.
|
|
Kinetic properties of double mutant S780A/K835G
In order to obtain an enzyme with high affinity to PEP and low sensitivity to allosteric inhibitors, a double mutant PEPC S780A/K835G was produced. The S0.5 value increased about 2-fold as compared with S780A, but it was still 40% of WT (Fig. 3A; Table 2). Sensitivity to malate and aspartate was almost completely lost, although the extent of slight inhibition was larger for malate than aspartate (Fig. 2). Measurement of activity at higher concentrations of the inhibitors up to 40 mM did not give I0.5 values for both inhibitors (Table 1). Although stronger desensitization was observed for S780A/K835G than K835G, this appears to be due to the difference in their S0.5 values. In fact, when the concentration of PEP was lowered to 0.5 mM for S780A/K835G so as to bring the extent of PEP saturation equal to K835G at 2 mM, the inhibition curve of S780A/K835G was almost superimposable on the curve of K835G (Fig. 2A, B). The sensitivities to allosteric activators, glucose 6-phosphate and glycine, were not affected by the introduced mutations (Tables 1, 2).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In combination with our previous X-ray crystallographic analysis (Kai et al., 1999), the present study using site-directed mutagenesis strongly suggests that the two conserved basic residues, Lys-835 and Arg-894 of maize C4-form PEPC, are involved in the binding with the allosteric inhibitors. The mutant enzymes, which had been desensitized to the negative feedback inhibitors but retained their catalytic activity, may be useful for a variety of metabolic engineering approaches. Although simultaneous desensitization to aspartate and malate was always observed by the replacement of a single amino acid residue, the degree of inhibition by malate was always larger than that of aspartate (Fig. 3). This tendency is presumably due to the interaction of malate at the catalytic site as a product analogue at higher concentrations.
Bläsing et al. (2000) showed that the S0.5 value for PEP decreased by about one-third when the C4-characteristic Ser-774 of a C4-form PEPC in F. trinervia was replaced with Ala which is conserved in non-C4-type PEPCs. It was also observed here with the maize C4-form PEPC it was also observed that the same replacement of the corresponding Ser-780 diminished the S0.5 value for PEP to one-quarter. Thus the role of this Ser was established for both dicot and monocot C4-form PEPCs.
By the introduction of a double mutation S780A/K835G, we succeeded in producing an enzyme with low S0.5 and high I0.5, demonstrating that these properties could be conferred simultaneously to PEPC. This mutant enzyme may be of use to make a C4-like cycle in C3 plants. To date, two different enzyme forms that would be less down-regulated and would show strong activity in plant cells have been used: one study exploited an engineered potato PEPC (Rademacher et al., 2002) and the other a PEPC from the cyanobacterium Synechococcus vulcanus (SvPEPC) (Chen et al., 2004). In the former case, the N-terminal truncated enzyme or the enzyme mutated at the regulatory phosphorylation site (Ser to Asp) was used. Both modifications resulted in enzymes with lowered sensitivity to malate inhibition. In the latter case, recombinant SvPEPC was malate/aspartate-insensitive in the normal cytosolic pH range (Chen et al., 2004). Although these modifications and protein sources were also available for metabolic engineering, maize PEPC was considered to be necessary for this technology, especially in applying it to rice. Since an extremely high level of expression was achieved in rice by the introduction of partial maize genomic DNA containing the gene for the C4-form PEPC (Miyao and Fukayama, 2003), this system will be quite useful for the expression of the engineered PEPC developed here. To construct the plasmid, a fragment (approximately 4 kb in size) of genomic DNA of the Ppc gene containing exons 8 and 9 where the mutation sites reside is subcloned and mutagenized by a conventional method, and then the mutagenized fragment is inserted back to the original plasmid.
Recently it was shown that, in rice, in which the maize PEPC was highly expressed, a nocturnal phosphorylation of PEPC was observed as an unexpected result, while the diurnal phosphorylation as in C4 plants was not (Fukayama et al., 2006). Because the malate-sensitivity of PEPC is altered by phosphorylation on its conserved Ser residue, the non-phosphorylated daytime form PEPC was thought to be less active in the transgenic rice. To avoid this problem, the phosphorylation-independent malate-insensitive feature is necessary for ideal PEPC functioning in rice C3 cell environments.
Figure 4 shows our present strategy for the enhancement of photosynthetic CO2 fixation in a C3 plant, tobacco. In scheme (A), the engineered maize C4-form PEPC and maize PEP carboxykinase (PCK) (Furumoto et al., 1999) are both expressed in the chloroplast, so as to convert HCO to CO2 at the expense of energy. Since Rubisco utilizes CO2 and not HCO as a substrate, an increase in CO2 supply to the microdomain around Rubisco would facilitate its CO2-fixation reaction. Scheme (B) shows a minimal C4-like pathway to be installed. Bicarbonate (HCO) in the cytoplasm is fixed onto PEP to produce oxaloacetate (OAA), and then OAA is transported into chloroplasts by a translocator, for example, a malate/Pi exchange translocator (Eastmond et al., 1997) or a dicarboxylate transporter (Jeong et al., 2004). In the chloroplasts, OAA is decarboxylated to supply CO2 to Rubisco by PCK. PEP formed in the chloroplasts is exported to the cytoplasm as the substrate for PEPC, by the action of a PEP/Pi translocator (Knappe et al., 2003). We are planning to introduce not only the enzymes but also the translocators into a C3 plant to complete the cycle. Although several systems of C4 photosynthesis in single cells have been envisaged and no successful improvement of photosynthetic productivity has been achieved yet, there still remains many trials to be done before this idea is abandoned (Mitchell and Sheehy, 2006). It should be emphasized that no work has yet been performed that introduced metabolite translocators to complete the C4-like cycle.
|
|
Acknowledgements |
|---|
This work was supported in part by grants-in-aid for Scientific Research (B) from the Ministry of Education, Science, Sports, and Culture of Japan and a Grant for the Recombinant Plant Project from the Ministry of Agriculture, Forestry and Fisheries of Japan. The authors are grateful to Miss Erika Akedo (Kinki University) for characterization of E. coli mutant PEPC, K773G, which showed only a slight activity in the cell extracts of F15/pT(K773G).
|
Footnotes |
|---|
* Present address: Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
These authors contributed equally to this work.
Present address: Department of Environmental and Biotechnological Frontier Engineering, Fukui University of Technology, 3-6-1, Gakuen, Fukui City, Fukui Prefecture 910-8505, Japan.
|
Abbreviations |
|---|
A0.5, concentration of an activator required for 50% of maximum activation; G6P, glucose 6-phophate; I0.5, concentration of an inhibitor required for 50% inhibition; PCK, phosphoenolpyruvate carboxykinase; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PEPC-k, phosphoenolpyruvate carboxylase kinase; S0.5, half-saturation concentration of substrate or metal cofactor; Vmax, maximum velocity.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bläsing OE, Westhoff P, Svensson P. Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. Journal of Biological Chemistry (2000) 275:27917–27923.
Bowes G, Rao SK, Estavillo GM, Reiskind JB. C4 mechanisms in aquatic angiosperms: comparisons with terrestrial C4 systems. Functional Plant Biology (2002) 29:379–392.[CrossRef][Web of Science]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Chen LM, Li KZ, Miwa T, Izui K. Overexpression of a cyanobacterial phosphoenolpyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis changes amino acid metabolism. Planta (2004) 219:440–449.[Web of Science][Medline]
Dong L, Hata S, Izui K. High-level expression of maize C4-type phosphoenolpyruvate carboxylase in Escherichia coli and its rapid purification. Bioscience, Biotechnology and Biochemistry (1997) 61:545–546.[Medline]
Dong LY, Masuda T, Kawamura T, Hata S, Izui K. Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays: comparison with the C4-form enzyme. Plant and Cell Physiology (1998) 39:865–873.
Dong L, Patil S, Condon SA, Haas EJ, Chollet R. The conserved C-terminal tetrapeptide of sorghum C4 phosphoenolpyruvate carboxylase is indispensable for maximal catalytic activity, but not for homotetramer formation. Archives of Biochemistry and Biophysics (1999) 371:124–128.[CrossRef][Web of Science][Medline]
Eastmond PJ, Dennis DT, Rawsthorne S. Evidence that a malate/inorganic phosphate exchange translocator imports carbon across the leucoplast envelope for fatty acid synthesis in developing castor seed endosperm. Plant Physiology (1997) 114:851–856.[Abstract]
Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudoh S, Furbank RT, Miyao M. Activity regulation and physiological impacts of the maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants. Photosynthesis Research (2003) 77:227–239.[CrossRef][Web of Science][Medline]
Fukayama H, Tamai T, Taniguchi Y, Sullivan S, Miyao M, Nimmo H. Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice. The Plant Journal (2006) 47:258–268.[CrossRef][Web of Science][Medline]
Furumoto T, Hata S, Izui K. cDNA cloning and characterization of maize phosphoenolpyruvate carboxykinase, a bundle sheath cell-specific enzyme. Plant Molecular Biology (1999) 41:301–311.[CrossRef][Web of Science][Medline]
Furumoto T, Izui K, Quinn V, Furbank RT, von Caemmerer S. Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis. Plant Physiology (2007) 144:1936–1945.
Häusler RE, Hirsch HJ, Kreuzaler F, Peterhansel C. Overexpression of C4-cycle enzymes in transgenic C3 plants: a biotechnological approach to improve C3-photosynthesis. Journal of Experimental Botany (2002) 53:591–607.
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene (1989) 77:51–59.[CrossRef][Web of Science][Medline]
Izui K, Matsumura H, Furumoto T, Kai Y. Phosphoenolpyruvate carboxylase: a new era of structural biology. Annual Review of Plant Biology (2004) 55:69–84.[CrossRef][Medline]
Jeong J, Suh S, Guan C, Tsay YF, Moran N, Oh CJ, An CS, Demchenko KN, Pawlowski K, Lee Y. A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family. Plant Physiology (2004) 134:969–978.
Kai Y, Matsumura H, Inoue T, Terada K, Nagara Y, Yoshinaga T, Kihara A, Tsumura K, Izui K. Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition. Proceedings of the National Academy of Sciences, USA (1999) 96:823–828.
Kai Y, Matsumura H, Izui K. Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms. Archives of Biochemistry and Biophysics (2003) 414:170–179.[CrossRef][Web of Science][Medline]
Knappe S, Lottgert T, Schneider A, Voll L, Flugge UI, Fischer K. Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis-AtPPT1 may be involved in the provision of signals for correct mesophyll development. The Plant Journal (2003) 36:411–420.[CrossRef][Web of Science][Medline]
Kogami H, Syono M, Koike T, Yanagisawa S, Izui K, Sentoku N, Tanifuji S, Uchimiya H, Toki S. Molecular and physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the control of the cauliflower mosaic virus 35S promoter. Transgenic Research (1994) 3:287–296.[CrossRef][Web of Science]
Matsumura H, Xie Y, Shirakata S, Inoue T, Yoshinaga T, Ueno Y, Izui K, Kai Y. Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. Structure (2002) 10:1721–1730.[Medline]
Matsuoka M, Furbank RT, Fukayama H, Miyao M. Molecular engineering of C4 photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology (2001) 52:297–314.[CrossRef][Web of Science][Medline]
Mitchell PL, Sheehy LE. Surcharging rice photosynthesis to increase yield. New Phytologist (2006) 171:688–693.[CrossRef][Web of Science][Medline]
Miyao M, Fukayama H. Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants. Archives of Biochemistry and Biophysics (2003) 414:197–203.[Web of Science][Medline]
Nimmo HG. Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plants. Archives of Biochemistry and Biophysics (2003) 414:189–196.[CrossRef][Web of Science][Medline]
O'Leary MH. Phosphoenolpyruvate carboxylase: an enzymologist's view. Annual Review of Plant Physiology (1982) 33:297–231.[Web of Science]
Rademacher T, Häusler RE, Hirsch HJ, Zhang L, Lipka V, Weier D, Kreuzaler F, Peterhänsel C. An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. The Plant Journal (2002) 32:25–39.[CrossRef][Web of Science][Medline]
Raines CA. Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle. Plant, Cell and Enviroment (2006) 29:331–339.[CrossRef]
Surridge C. Agricultural biotech: the rice squad. Nature (2002) 416:576–578.[CrossRef][Medline]
Svensson P, Bläsing OE, Westhoff P. Evolution of C4 phosphoenolpyruvate carboxylase. Archives of Biochemistry and Biophysics (2003) 414:180–188.[CrossRef][Web of Science][Medline]
Takahashi-Terada A, Kotera M, Ohshima K, Furumoto T, Matsumura H, Kai Y, Izui K. Maize phosphoenolpyruvate carboxylase: mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation. Journal of Biological Chemistry (2005) 12:11798–11806.
Terada K, Fujita N, Katsuki H, Izui K. Construction of a plasmid for high level expression of Escherichia coli phosphoenolpyruvate carboxylase. Bioscience, Biotechnology and Biochemistry (1995) 59:735–737.[Medline]
Terada K, Izui K. Site-directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase: His 138 is essential for the second partial reaction. European Journal of Biochemistry (1991) 202:797–803.[Web of Science][Medline]
Tsuchida Y, Furumoto T, Izumida A, Hata S, Izui K. Phosphoenolpyruvate carboxylase kinase involved in C4 photosynthesis in Flaveria trinervia: cDNA cloning and characterization. FEBS Letters (2001) 507:318–322.[CrossRef][Web of Science][Medline]
Vidal J, Chollet R. Regulatory phosphorylation of C4 PEP carboxylase. Trends in Plant Science (1997) 2:230–237.[CrossRef][Web of Science]
von Caemmerer S. C4 photosynthesis in a single C3 cell is theoretically inefficient but may ameliorate internal CO2 diffusion limitations of C3 leaves. Plant, Cell and Environment (2003) 26:1191–1197.[CrossRef]
Yano M, Terada K, Umiji K, Izui K. Catalytic role of an arginine residue in the highly conserved and unique sequence of phosphoenolpyruvate carboxylase. Journal of Biochemistry (1995) 11:1196–1200.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||