Abstract
The Calvin-Benson-Bassham (CBB) cycle is responsible for CO2 assimilation and carbohydrate production in oxyphototrophs. Phosphoribulokinase (PRK) is an essential enzyme of the CBB cycle in photosynthesis, catalyzing ATP-dependent conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate. The oxyphototrophic PRK is redox-regulated and can be further regulated by reversible association with both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidized chloroplast protein CP12. The resulting GAPDH/CP12/PRK complex is central in the regulation of the CBB cycle; however, the PRK-CP12 interface in the recently reported cyanobacterial GAPDH/CP12/PRK structure was not well resolved, and the detailed binding mode of PRK with ATP and Ru5P remains undetermined, as only apo-form structures of PRK are currently available. Here, we report the crystal structures of cyanobacterial (Synechococcus elongatus) PRK in complex with ADP and glucose-6-phosphate and of the Arabidopsis (Arabidopsis thaliana) GAPDH/CP12/PRK complex, providing detailed information regarding the active site of PRK and the key elements essential for PRK-CP12 interaction. Our structural and biochemical results together reveal that the ATP binding site is disrupted in the oxidized PRK, whereas the Ru5P binding site is occupied by oxidized CP12 in the GAPDH/CP12/PRK complex. This structure-function study greatly advances the understanding of the reaction mechanism of PRK and the subtle regulations of redox signaling for the CBB cycle.