Structural Basis of the Regulation of Carbon Fixation

The Calvin-Benson cycle is the metabolic pathway for carbon fixation in plants, algae and cyanobacteria. The pathway takes carbon dioxide (the most oxidized form of carbon) and converts it into sugars, which are fed into metabolism. The pathway contains 13 enzymatic steps in 3 stages: carbon fixation, carbon reduction and substrate regeneration. As this process is energy intensive and uses ATP and NAPDPH, it is tightly regulated.

A central regulation mechanism of the Calvin Benson cycle is via the redox state. The change in state of the cytoplasm allows for the control of key enzymes in the Calvin Benson cycle, including glyceradehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). GAPDH catalyses the reduction of 1,3-bisphosphoglycerate to glycerate-3-phosphate, with NADPH as the electron donor. PRK is essential for the regeneration of the Calvin Benson cycle and catalyses the ATP-dependent phosphorylation of ribulose-5-phosphate to ribulose 1,5-bisphosphate.

In the light, the cytoplasm becomes reduced as photosystem I produces reduced ferredoxin. In the dark, or under other stress conditions, the cytoplasm is oxidised. The redox change enables a small regulatory protein called CP12 to form an inhibitory complex with GAPDH and PRK in the dark, mediated by two pairs of disulfide-forming cysteines at the N- and C-termini of CP12. Stanley et al (2013) identified the diverse role of CP12 regulation in response to light but also to temperature, nitrogen deprivation and osmotic stress. More recently, McFarlane et al (2019) published a cryo-EM structure of the GAPDH-CP12-PRK complex and crystal structures of CP12GAPDH from a thermophilic cyanobacterium, which provided detailed structural information on the interaction of the partner proteins.

This project will build on existing knowledge of the GAPDH-CP12-PRK complex and will extend structural understanding of complex formation using X-ray crystallography and cryo-EM. Previous research has focused on cyanobacteria however, research from Yu et al (2020) has highlighted the possibility of investigating the plant complex. We wish to elucidate relevant structures from economically important crops such as wheat and with different plant isoforms of CP12. Mutagenesis and biochemical experiments will be used to probe the structural understanding of the complex formation, in particular the role of the conserved C-terminal disulfide bond in PRK, which is of unknown function.

This research will provide greater understanding of the regulation of photosynthesis in plants. The regulatory mechanisms are likely to be conserved amongst different higher plants and this research will aid in developing plants with a higher carbon fixation efficiency.



McFarlane, C.R., Shah, N.R., Kabasakal, B.V., Echeverria, B., Cotton, C.A.R., Bubeck, D., and Murray, J.W. (2019). Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria. Proc. Natl. Acad. Sci. 116, 20984-20990.



Stanley, D.N., Raines, C.A., and Kerfeld, C.A. (2013). Comparative Analysis of 126 Cyanobacterial Genomes Reveals Evidence of Functional Diversity Among Homologs of the Redox-Regulated CP12 Protein1[C][W]. Plant Physiol. 161, 824-835.



Yu, A., Xie, Y., Pan, X., Zhang, H., Cao, P., Su, X., Chang, W., and Li, M. (2020). Photosynthetic Phosphoribulokinase Structures: Enzymatic Mechanisms and the Redox Regulation of the Calvin-Benson-Bassham Cycle. Plant Cell 32, 1556-1573.