THE IMPACT OF PHOSPHOMIMICS ON THE ALLOSTERIC REGULATION OF PHOSPHOENOLPYRUVATE CARBOCYATION (PEPC) FROM SETARIA VIRIDIS

Monipak Fidelia Lare

The carbon concentrating mechanism of C4 photosynthesis has been extensively studied, revealing a complex suite of anatomical and biochemical adaptations required for its high photosynthetic efficiency. While there is growing interest in optimizing this pathway to enhance crop resilience under abiotic stress, many questions remain regarding the regulation and evolution of its primary rate-limiting enzymes, particularly phosphoenolpyruvate carboxylase (PEPC). To support bioengineering efforts aimed at improving carbon assimilation, it is essential to better understand how specific factors influence PEPC kinetics and regulation.This thesis investigates how PEPC kinetic properties and allosteric regulation is fine-tuned through post-translational modifications in the C4 model species Setaria viridis. Focusing on phosphorylation at the conserved N-terminal Ser-11 residue, I explore how phosphomimic substitutions (SvPEPC_S11D and SvPEPC_S11E) at this serine residue impact PEPC kinetic parameters, including substrate affinity (Kₚₑₚ) and sensitivity to key allosteric regulators such as malate and glucose 6-phosphate (G6-P). These findings demonstrate that phosphorylation at Ser-11 plays a key role in modulating the trade-off between PEPC activity and feedback inhibition in C4 photosynthesis. Phosphomimic variants at Ser-11 were generated by substituting this residue with the negatively charged aspartic acid (SvPEPC_S11D) or glutamic acid (SvPEPC_S11E). These PEPC variants had significantly different affinity for PEP (higher KPEP) compared to the wild-type enzyme (SvPEPC_WT), indicating the phosphomimics altered the PEP binding affinity. Additionally, both phosphomimics showed a higher AC50 for G6-P, suggesting enhanced sensitivity to this allosteric activator, while exhibiting elevated IC50 values for malate, indicating reduced susceptibility to feedback inhibition. Caution should be taken when interpreting the AC50 and IC50 data as much higher concentrations of the SvPEPC_WT enzyme were used in these assays compared to SvPEPC_S11D and SvPEPC_S11E enzymes. Together, these shifts in kinetic and allosteric parameters suggest that the phosphomimics do modify PEPC similarly to phosphorylation at Ser-11. However, there were differences between the response of SvPEPC_S11D and SvPEPC_S11E variants to G6-P and malate. The allosteric regulation of the SvPEPC_WT variant from the E. coli expression system also differed from the WT PEPC extracted from leaf tissue. This work provides direct evidence that the phosphomimic in the E. coli expression system have some but limited use in understanding the role of phosphorylation at Ser-11. Additionally, this work demonstrates that PEPC extracted from leaf tissue differs significantly from the enzyme generated in E. coli suggesting the potential importance of other post-translational modifications in PEPC responses to allosteric regulation.

THE IMPACT OF PHOSPHOMIMICS ON THE ALLOSTERIC REGULATION OF PHOSPHOENOLPYRUVATE CARBOCYATION (PEPC) FROM SETARIA VIRIDIS

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