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Flaveria is a genus of plants in the sunflower family (Asteraceae). Within the close species of Flaveria, there are several different photosynthetic metabolisms including C3, C4 and C3-C4 mixed metabolism. In C4 metabolism, phosphoenolpyruvate carboxylase (PEPC) catalyses the primary fixation of atmospheric CO2. In order to elucidate the discrete steps in PEPC evolution computational analysis was made for the PEPC protein sequences of C3, C3-C4 and C4 species of the dicot genus Flaveria. The predicted key amino acid residue changes and putative phosphorylation sites can advance our knowledge on plant photosynthesis metabolism, especially on the regulation of PEPC activity. One of the most notable amino acid residue changes found at 123 contains serine in C4 Flaveria species and occupied by arginine in C3, C3-C4, and C4 like Flaveria species and also the serine residue at this position was predicted as putative phosphorylation site. Functional expression and characterization of the C3, C3-C4 intermediate and C4 PEPC of Flaveria species enzymes can reveal that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence similarity.