Maize C₄-form phosphoenolpyruvate carboxylase engineered to be functional in C₃ plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity

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 carbox...

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Published in:Journal of experimental botany 2008, Vol.59 (7), p.1811-1818
Main Authors: Endo, Tatsuya, Mihara, Yuko, Furumoto, Tsuyoshi, Matsumura, Hiroyoshi, Kai, Yasushi, Izui, Katsura
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
C4 photosynthesis
Enzyme Inhibitors
Escherichia coli - metabolism
Genetic Complementation Test
genetic engineering
Genetic Engineering - methods
Kinetics
Mutation
PEP carboxylase
Phosphoenolpyruvate Carboxylase - antagonists & inhibitors
Phosphoenolpyruvate Carboxylase - genetics
Phosphoenolpyruvate Carboxylase - metabolism
Photosynthesis - genetics
Photosynthesis - physiology
site-directed mutagenesis
Substrate Specificity
Zea mays
Zea mays - enzymology
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Summary: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 4.1.1.31) 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.
ISSN:0022-0957
1460-2431
DOI:10.1093/jxb/ern018