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.
Highlights
Oxygenic phototrophs such as cyanobacteria, algae, and land plants convert carbon dioxide and water into carbohydrates and release the by-product oxygen, a process that can be divided into the light reactions and the Calvin-Benson-Bassham (CBB) cycle
We solved the structure of SePRK in complex with ADP and G6P and generated a docked model of SePRK bound with ATP and Ru5P based on our crystal structure
Since the two Ser residues are located near the g-phosphate group of ATP in our docked model, it is possible that they facilitate the proper orientation of ATP, especially its g-phosphate group
Summary
Oxygenic phototrophs such as cyanobacteria, algae, and land plants convert carbon dioxide and water into carbohydrates and release the by-product oxygen, a process that can be divided into the light reactions and the Calvin-Benson-Bassham (CBB) cycle (light-independent reactions). The CBB cycle is regulated by light/dark transitions through the redox states of the chloroplast stroma (Buchanan, 1980; Scheibe, 1991; Geiger and Servaites, 1994). PRK from oxygenic phototrophs differs significantly from RsPRK in primary sequence, oligomeric state, and regulatory mechanism (Buchanan, 1980; Tabita, 1980). In contrast to the octameric RsPRK, PRKs from oxygenic phototrophs are commonly presented as homodimers, and each monomer contains a pair of Cys residues at the N-terminal region. PRKs from plants and eukaryotic algae are redox-regulated through reversible reduction and oxidation of this Cys pair (Latzko et al, 1970; Wirtz et al, 1982; Milanez et al, 1991; Brandes et al, 1996), which is absent in RsPRK. Cyanobacterial PRKs contain a similar Cys pair at the N-terminal
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