Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin–Benson cycle

Abstract

In land plants and algae, the Calvin–Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central β-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes—the clamp loop—which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

Temperature Sensitive Photosynthesis: Point Mutated CEF-G, PRK, or PsbO Act as Temperature-Controlled Switches for Essential Photosynthetic Processes

“…Oxidation and reduction of this bridge is mediated by thioredoxin and results in deactivation and activation of PRK respectively. The flexibility of the "clamp loop" is thought to facilitate these conformational changes (Thieulin-Pardo et al, 2015;Gurrieri et al, 2019). The mutation in TSP2 may have interacted with this mechanism and interfered with the activation of PRK at high temperature.…”

New Light on Chloroplast Redox Regulation: Molecular Mechanism of Protein Thiol Oxidation

“…Our understanding of CBC redox regulation is even now growing. For example, its molecular details and evolutionary traits have been further elucidated by recent structural studies (FBPase and SBPase from Physcomitrella patens , Gütle et al, 2016; PRK from Chlamydononas reinhardtii and Arabidopsis thaliana , Gurrieri et al, 2019).…”

Crystal structure of chloroplastic thioredoxin z defines a novel type-specific target recognition

“…While CrPRK is a well-known CrTRXf2 target, presumably because of its partially negative surface around C55, another large positive patch is also present near C55 at the enzymatic pocket [48, 54]. The mixed environment of CrPRK C55 suggests an independent, alternative mechanism of redox activation by TRXz.…”

“…Proteomic analyses based on affinity purification chromatography and in vitro reconstitution of the cytosolic TRX system allowed the identification of 1053 TRX targets and 1052 putative regulatory sites in Chlamydomonas [35, 47]. Among these, all CBC enzymes were identified indicating that they are potential TRX interactors, and direct TRX-dependent activation of CBC enzymes was only demonstrated for PRK [48], FBPase [49], SBPase [50] and phosphoglycerate kinase (PGK) [51]. By contrast with land plants, photosynthetic GAPDH from Chlamydomonas reinhardtii (CrGAPDH) is not directly regulated by TRX but only indirectly through formation of the (A 4 -GAPDH) 2 -CP12 4 -PRK 2 complex [52–54].…”

“…By contrast with land plants, photosynthetic GAPDH from Chlamydomonas reinhardtii (CrGAPDH) is not directly regulated by TRX but only indirectly through formation of the (A 4 -GAPDH) 2 -CP12 4 -PRK 2 complex [52–54]. Among the five TRX types present in chloroplasts, only TRXf has been systematically compared to other types and demonstrated to target FBPase [21, 32], PRK [48, 54], GAPDH in land plants [54] and PGK in Chlamydomonas [51]. Structural analysis revealed that electrostatic complementarity was the principal driver of TRXf-targets recognition [55–59].…”