Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle and shed light on their molecular evolution in eukaryotes

Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that can later be released to fuel the organisms' activities. This chemical energy is stored in carbohydrate molecules, such as sugars, which are synthesized from carbon dioxide and water releasing oxygen as a waste product. In plants, algae and cyanobacteria, long-term energy storage in the form of sugars is produced by a subsequent sequence of light-independent (dark) reactions called the Calvin-Benson (CB) cycle. 
The CB cycle consists of 13 distinct reactions catalysed by 11 enzymes that are differentially regulated to coordinate the two stages (figure 1) of photosynthesis—electron transport and carbon fixation—and to couple them with the continuous changes in environmental light.
In such cycle atmospheric carbon dioxide is incorporated into already existing organic carbon compounds, such as ribulose bisphosphate (RuBP). Using the ATP and NADPH produced by the light-dependent reactions (lightside), the resulting compounds are then reduced and removed to form further carbohydrates, such as glucose. 
Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages (light-dependent and light-independent) of photosynthesis through a thiol-based mechanism.
 

Figure 1.    Photosynthesis takes place in two stages: light dependent reactions and the Calvin cycle. Light-dependent reactions (left side), which take place in the thylakoid membrane, use light energy to make ATP and NADPH. In the chloroplast, the Calvin cycle (right side) uses energy derived from these compounds to make Glyceraldehyde 3-phosphate (GA3P) from CO2. (*) Figure adapted from https://courses.lumenlearning.com

TRXs reduce disulfide bonds in target proteins, controlling the redox state and modulating the activity of target enzymes. Different classes of TRXs are known (i.e., TRX-f, -m, -x, -y, and -z). They are ancient and strongly evolutionarily conserved.
Among the11 enzymes of the CB cycle, the TRXs target phosphoribulokinase (PRK) had notably still to be characterizedat the atomic scale. To accomplish this goal and complete the redoxstructural proteome of the CB cycle, authors determined at the XRD1 beamline at Elettra the crystal structures of PRK from twomodel species: the green alga Chlamydomonas reinhardtii(CrPRK) and the land plant Arabidopsisthaliana(AtPRK). The PRK three-dimensional structure (figure 2) shows the molecule to be an elongated homodimer characterized by a large central β-sheet of 18strands, 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 groupsof substrates – and an exposed negative region to attract a positively charged class of TRXs (TRX-f), justifying experimental evidences between  different TRX classes (TRX-m versus TRX-f2) in the capability of the reductive activation of PRKs.

Figure 2.  The crystal structure of photosynthetic PRK. Representation of the overall crystal structure of reduced phosphoribulokinase (PRK) from Chlamydomonas reinhardtii.  The clamp loop is highlighted in orange. Each monomer contains two pairs of cysteines, one in the active site and one in the dimer interface close to the C-terminal end of the protein chain. Cysteine residues are indicated and represented as sticks. CrPRK binds two sulphate ions (one for each monomer), represented as spheres, from the crystallization solution. Arg64, represented as sticks, is one of the residues stabilizing the anions.
 

The inspection of the catalytic cavity revealed that the regulatory cysteines can be found 13 Å apart and connected by a flexible region exclusive tophotosynthetic eukaryotes—the clamp loop—which is believed to be essential for oxidation-induced structural rearrangements.
Very interestingly, structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and anincreased number of random coiled regions, suggesting that a general loss in structural rigiditycorrelates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

This research was conducted by the following research team:

Libero Gurrieri¹, Mirko Zaffagnini¹, Paolo Trost¹, Francesca Sparla¹, Alessandra Del Giudice², Nicolae Viorel Pavel², Nicola Demitri³, Maurizio Polentarutti³, Giuseppe Falini⁴, Simona Fermani⁴, Pierre Crozet⁵, Christophe H. Marchand⁵, Julien Henri⁵, Stéphane D. Lemaire⁵

¹ Department of Pharmacy and Biotechnology – FaBiT, University of Bologna, Bologna, Italy
² Department of Chemistry, University of Rome ‘Sapienza’, Rome, Italy
³ Elettra - Sincrotrone Trieste S.C.p.A., Trieste, Italy
⁴ Department of Chemistry ‘G. Ciamician’, University of Bologna, Bologna, Italy
⁵ Institut de Biologie Physico-Chimique, UMR8226, CNRS, Sorbonne Université, Paris, France

Contact persons:

Maurizio Polentarutti e-mail: