Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria:: determinants for high tolerance towards the inhibitor L-malate

During the evolution of angiosperms, C(4) phosphoenolpyruvate carboxylases have evolved several times independently from ancestral non-photosynthetic isoforms. They show distinct kinetic and regulatory properties when compared with the C(3) isozymes. To identify the evolutionary alterations which are responsible for C(4)-specific properties, particularly the increased tolerance towards the allosteric inhibitor L-malate, the photosynthetic phosphoenolpyruvate carboxylase of Flaveria trinervia Mohr (C(4)) and its ortholog from the closely related C(3) plant Flaveria pringlei Gand. were examined using reciprocal enzyme chimeras. The main determinants for a high tolerance towards L-malate were located in the C-terminal region of the C(4) enzyme. The effect of interchanging the region between amino acids 296 and 437 was strongly dependent upon the activation of the enzyme by glucose-6-phosphate. This confirms earlier observations that this region is important for the regulation of the enzyme by glucose-6-phosphate and that it harbours determinants for the different response of the C(3) and the C(4) enzyme towards this allosteric activator. In addition, it was possible to demonstrate that the only C(4)-specific amino acid, a serine in the C-terminal part of the enzyme, is not involved in conferring an increased L-malate tolerance to the C(4) enzyme.