Comparative enzymology of the convergent co-option of phosphoenolpyruvate carboxylase for C4 photosynthesis

Approximately a third of the world population relies on rice every day; however, increases rice yields are not increasing to match the predicted increase in world population. \nEngineering the C4 photosynthetic carbon dioxide concentrating mechanism in rice may reduce inefficiencies in rice and increase yield. C4 photosynthesis uses the enzyme \nphosphoenolpyruvate carboxylase (PEPC) to fix atmospheric carbon dioxide in a four carbon acid which is shuttled to a separate compartment where the carbon dioxide is \nreleased and concentrated around the key enzyme of the Calvin-Benson cycle. Plants that have evolved the C4 cycle co-opted PEPC from a background role. The expression patterns and kinetic properties of PEPC are adapted to the demands of carbon fixation. The changes in gene expression have been studied previously through high-throughput sequencing techniques but the biochemical changes remain largely unexplored. Understanding how PEPC adapted is a key part of engineering C4 photosynthesis. In this thesis I looked at the kinetic changes in C4 PEPC from one of the youngest C4 species in the genus Flaveria which showed that the C4 PEPC has a higher specificity for bicarbonate, a lower specificity for phosphoenolpyruvate, and a decreased sensitivity to inhibitors when compared to the non-C4 \nFlaveria PEPC. I then compared the kinetic properties of PEPCs from species in the genus Panicum, an early and successful C4 origin. Comparison showed a convergence in kinetic properties of C4 PEPCs in Panicum and Flaveria. However, the changes seen in C4 Panicum PEPC are quantitively greater showing further adaptation. C4 specific changes resulting from specific amino acids changes were investigated. It was shown that the same C4 specific \nmutation was responsible for similar reduction in magnitude of PEP specificity in both Flaveria and Panicum C4 PEPC. Other investigated C4 specific amino acids were shown not \nto contribute to major kinetic properties. This surprising result suggested other selection forces act in the evolution of C4 PEPC. The evolution of C4 PEPC involved adaption \nincrease in the specificity for bicarbonate. It was shown the that bicarbonate plays a part in the evolution of PEPC but adaptations towards inhibitor and PEP specificity are selected preferentially.