JXB Advance Access originally published online on April 11, 2008
Journal of Experimental Botany 2008 59(7):1811-1818; doi:10.1093/jxb/ern018
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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
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
* 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.
Received 3 December 2007; Revised 6 January 2008 Accepted 11 January 2008