Allosteric Regulation and Kinetic Parameters of a Key Enzyme Driving the Initial Carboxylation of C4 Photosynthesis

Ryan Lee Wessendorf

doi:10.7273/000005092

The carbon concentrating mechanism of C4 photosynthesis has been extensively studied over the last 35 years and there has been many discoveries pertaining to the complex set of anatomical and biochemical adaptions required for its efficient operation. Although there is growing interest in optimizing this pathway to alleviate abiotic stress on food crops, there are many questions remaining about the evolution and allosteric regulation of primary rate-limiting enzymes of this mechanism, such as phosphoenolpyruvate carboxylase. Therefore, detailed analysis of amino acid modifications that control kinetic trade-offs and their relationship to carbon capture of atmospheric gasses is necessary to advance such breeding efforts. This dissertation describes some of the genetic and biochemical factors that control CO2 flux into the C4 pathway. First, two grass species, Oropetium thomaeum and Paspalum vaginatum, were used to characterize single amnio acid and region substitutions as they relate to variation in kinetic trade-offs. Although many factors may contribute to variation in kinetic trade-offs, we hypothesized that region II and residue 353 can be manipulated in phosphoenolpyruvate carboxylase and likely drive variation in allosteric regulation and substrate affinity, supporting previous suggestions that the evolution of specific amino acids can be used to bioengineer a more optimal food crops. Second, these diverse isoforms of grass phosphoenolpyruvate carboxylase were used to identify cooperation of multiple amino acids that may drive variation in kinetic trade-offs. No cooperation was observed, but the 780 residue was found to drive a substrate trade-off. These results highlighted the importance of future work on kinetic trade-offs and demonstrate that a larger more diverse collection of phosphoenolpyruvate carboxylase enzymes could unlock a better general understanding of kinetic trade-offs. Finally, kinetic trade-offs were explored across a larger selection of C4 grass species. We observed individual kinetic trade-off responses, but these responses were not consistent across a larger panel of C4 species. Furthermore, we connected variation in allosteric regulation of one substrate to overall fitness of phosphoenolpyruvate carboxylase. Overall, this dissertation advances the understanding of evolutionary substitutions to amino acid composition that drives kinetic trade-offs that impact overall fitness of a rate-limiting enzyme driving C4 photosynthesis.

Allosteric Regulation and Kinetic Parameters of a Key Enzyme Driving the Initial Carboxylation of C4 Photosynthesis

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