Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C-4 photosynthesis. It has evolved from ancestral non-photosynthetic (C-3) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C-4 PEPCases were adapted to their new function in C-4 photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C-4 plant Alternanthera pungens H.B.K., the C-3/C-4 intermediate plant A. tenella Colla, and the C-3 plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K (0.5) PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C-4) as a C-4 PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C-3) and A. tenella (C-3/C-4) were found to be typical C-3 PEPCase isozymes. The C-4 characteristics of the PEPCase of A. pungens were accompanied by the presence of the C-4-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C-4 PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3' untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.

C4 phosphoenolpyruvate carboxylases have evolved several times independently from non‐photosynthetic C3 ancestral enzymes. To identify C4‐specific determinants at the amino acid level the two orthologous ppcA PEPCases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were used as a model system. In a previous publication [Bläsing et al., J. Biol. Chem. 275 (2000) 27917–27923] it was reported that the serine at position 774 is an invariant residue in all C4 phosphoenolpyruvate carboxylases. Here we show by swapping experiments and site‐directed mutagenesis that the serine 774 and amino acids 296–437 explain two thirds of the C4 characteristic phosphoenolpyruvate saturation kinetics when investigated in the C3 background. In addition, the results indicate that the determinants functionally interact with each other.

C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.

C 4 phosphoenol pyruvate ( P ‐pyruvate) carboxylases have evolved from ancestral C 3 P ‐pyruvate carboxylases during the evolution of C 4 photosynthesis (Lepiniec et al., 1994). To meet the requirements of a new metabolic pathway, the C 4 enzymes have gained distinct kinetic and regulatory properties compared to C 3 enzymes. Our interest is to deduce the structure responsible for these C 4 ‐specific properties. As a model system, the orthologous ppcA P ‐pyruvate carboxylases of Flaveria trinervia (C 4 ) and Flaveria pringlei (C 3 ) were investigated by expressing them in Escherichia coli using their cDNAs. The K m ( P ‐pyruvate) was about ten times higher for the C 4 enzyme (650 μM) than for the C 3 enzyme (60 μM). The activation by glucose 6‐phosphate, which was shown by a decrease in the K m ( P ‐pyruvate), was about twice for the C 4 enzyme and three times for the C 3 enzyme. The C 3 enzyme showed a very high sensitivity to L‐malate with an I 0.5 (50% inhibition) value of 80 μM malate, whereas the C4 enzyme was much less sensitive with a I 0.5 value of 1.2mM malate. To locate the structural positions responsible for these differences in kinetic and regulatory properties, chimeras of these 95% identical enzymes were made. In this study, the first 437 residues of the 966‐amino‐acid protein were interchanged. The results showed that the N‐terminal part of the enzyme was responsible for a small but significant part of the kinetic difference observed between these two isoenzymes. Additionally, the results suggest that the N‐terminus was the site for glucose 6‐phosphate activation and was also responsible for the observed difference in activation by this sugar phosphate. The difference in inhibition by L‐malate, however, is suggested to originate mainly from the C‐terminal part of the enzyme.