title: Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity.
abstract: 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. The resulting mutant enzymes were active but their sensitivities to the inhibitors were greatly diminished. Secondly, a Ser residue (S780) characteristically conserved in all C(4)-form PEPC was replaced by Ala conserved in C(3)- and root-form PEPCs to decrease the half-maximal concentration (S(0.5)) of PEP. The double mutant enzyme (S780A/K835G) showed diminished sensitivity to malate and decreased S(0.5)(PEP) with equal maximal catalytic activity (V(m)) to the wild-type PEPC, which will be quite useful as a component of the C(4)-like pathway to be introduced into C(3) plants.
authors.affiliation: Laboratory of Plant Physiology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Endo
authors.forename: Yuko
authors.initials: Y
authors.lastname: Mihara
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Furumoto
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kai
authors.forename: Katsura
authors.initials: K
authors.lastname: Izui
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005818:Genetic Engineering / Q000379:methods*; D007700:Kinetics; D009154:Mutation; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D010788:Photosynthesis / Q000235:genetics* / Q000502:physiology; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D010730:Phosphoenolpyruvate Carboxylase