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 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 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 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 C 4-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. © The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.