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CP12 is a redox-dependent conditionally disordered protein universally distributed in oxygenic photosynthetic organisms. It is primarily known as a light-dependent redox switch regulating the reductive step of the metabolic phase of photosynthesis. In the present study, a small angle X-ray scattering (SAXS) analysis of recombinant Arabidopsis CP12 (AtCP12) in a reduced and oxidized form confirmed the highly disordered nature of this regulatory protein. However, it clearly pointed out a decrease in the average size and a lower level of conformational disorder upon oxidation. We compared the experimental data with the theoretical profiles of pools of conformers generated with different assumptions and show that the reduced form is fully disordered, whereas the oxidized form is better described by conformers comprising both the circular motif around the C-terminal disulfide bond detected in previous structural analysis and the N-terminal disulfide bond. Despite the fact that disulfide bridges are usually thought to confer rigidity to protein structures, in the oxidized AtCP12, their presence coexists with a disordered nature. Our results rule out the existence of significant amounts of structured and compact conformations of free AtCP12 in a solution, even in its oxidized form, thereby highlighting the importance of recruiting partner proteins to complete its structured final folding.

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an α8β8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.

International audience; Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin–Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX–target recognitions.

Oxygenic phototrophs use the Calvin–Benson cycle to fix CO2 during photosynthesis. In the dark, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two enzymes of the Calvin–Benson cycle, form an inactive complex with the regulatory protein CP12, mainly under the control of thioredoxins and pyridine nucleotides. In the light, complex dissociation allows GAPDH and PRK reactivation. The GAPDH/CP12/PRK complex is conserved from cyanobacteria to angiosperms and coexists in land plants with an autoassembling GAPDH that is analogously regulated. With the recently described 3D structures of PRK and GAPDH/CP12/PRK, the structural proteome of this ubiquitous regulatory system has been completed. This outcome opens a new avenue for understanding the regulatory potential of photosynthetic carbon fixation by laying the foundation for its knowledge-based manipulation.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant thioredoxins evolved into seven types with diverse subcellular localization and distinct protein targets selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the first crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows TRXz with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. TRXz distinct electronegative surface thereby extends the repertoire of TRX-target recognitions.

Water shortage is an increasing problem affecting crop yield. Accumulation of compatible osmolytes is a typical plant response to overcome water stress. Sucrose synthase 1 (SUS1), and glucan, water dikinase 2 (GWD2) and &delta, 1-pyrroline-5-carboxylate synthetase 1 (P5CS1) are members of small protein families whose role in the response of Arabidopsis thaliana plants to mild osmotic stress has been studied in this work. Comparative analysis between wild-type and single loss-of-function T-DNA plants at increasing times following exposure to drought showed no differences in the content of water-insoluble carbohydrate (i.e., transitory starch and cell wall carbohydrates) and in the total amount of amino acids. On the contrary, water-soluble sugars and proline contents were significantly reduced compared to wild-type plants regardless of the metabolic pathway affected by the mutation. The present results contribute to assigning a physiological role to GWD2, the least studied member of the GWD family, strengthening the involvement of SUS1 in the response to osmotic stress, showing a greater contribution of soluble sugars than proline in osmotic adjustment of Arabidopsis in response to drought. Finally, an interaction between proline and soluble sugars emerged, albeit its nature remains speculative and further investigations will be required for complete comprehension.

Culturing microalgae using dairy-wastes offers the opportunity of producing valuable biomass for different industrial applications. The capability of four Chlorella species and a recombinant Chlamydomonas reinhardtii strain to mixotrophically grow in wastewaters from an Italian dairy factory was investigated. A robust algal growth could be efficiently sustained in these wastes, despite the abundance of d -Lactose (~4% w/v), that could not be metabolized by any microalgal species. Non axenic cocultivation of microalgae together with microbial communities from the dairy wastes resulted in a marked decrease of their pollution load, thus reducing the necessity of expensive treatments before their discharge in the municipal sewage system. Microalgae cultivated using dairy-wastes were characterized by a lipid content ranging from 12% to 21% (w/w), with Auxenochlorella protothecoides reaching the highest lipid productivity (~0.16 g/L/d) whereas the transplastomic C. reinhardtii strain expressing a thermostable β-glucosidase reached a recombinant enzyme productivity of 0.18 mg/L/d.

International audience; Thioredoxins (TRXs) are major protein disulfide reductases of the cell. Their redox activity relies on a conserved Trp-Cys-(Gly/Pro)-Pro-Cys active site bearing two cysteine (Cys) residues that can be found either as free thiols (reduced TRXs) or linked together by a disulfide bond (oxidized TRXs) during the catalytic cycle. Their reactivity is crucial for TRX activity, and depends on the active site microenvironment. Here, we solved and compared the 3D structure of reduced and oxidized TRX h1 from Chlamydomonas reinhardtii (CrTRXh1). The three-dimensional structure was also determined for mutants of each active site Cys. Structural alignments of CrTRXh1 with other structurally solved plant TRXs showed a common spatial fold, despite the low sequence identity. Structural analyses of CrTRXh1 revealed that the protein adopts an identical conformation independently from its redox state. Treatment with iodoacetamide (IAM), a Cys alkylating agent, resulted in a rapid and pH-dependent inactivation of CrTRXh1. Starting from fully reduced CrTRXh1, we determined the acid dissociation constant (pK a) of each active site Cys by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analyses coupled to differential IAM-based alkylation. Based on the diversity of catalytic Cys deprotonation states, the mechanisms and structural features underlying disulfide redox activity are discussed.

Thioredoxins (TRXs) are major protein disulfide reductases of the cell. Their redox activity relies on a conserved Trp-Cys-(Gly/Pro)-Pro-Cys active site bearing two cysteine (Cys) residues that can be found either as free thiols (reduced TRXs) or linked together by a disulfide bond (oxidized TRXs) during the catalytic cycle. Their reactivity is crucial for TRX activity, and depends on the active site microenvironment. Here, we solved and compared the 3D structure of reduced and oxidized TRX h1 from Chlamydomonas reinhardtii (CrTRXh1). The three-dimensional structure was also determined for mutants of each active site Cys. Structural alignments of CrTRXh1 with other structurally solved plant TRXs showed a common spatial fold, despite the low sequence identity. Structural analyses of CrTRXh1 revealed that the protein adopts an identical conformation independently from its redox state. Treatment with iodoacetamide (IAM), a Cys alkylating agent, resulted in a rapid and pH-dependent inactivation of CrTRXh1. Starting from fully reduced CrTRXh1, we determined the acid dissociation constant (pKa) of each active site Cys by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry analyses coupled to differential IAM-based alkylation. Based on the diversity of catalytic Cys deprotonation states, the mechanisms and structural features underlying disulfide redox activity are discussed.

The genome of Arabidopsis thaliana encodes three glucan, water dikinases. Glucan, water dikinase 1 (GWD1; EC 2.7.9.4) and phosphoglucan, water dikinase (PWD; EC 2.7.9.5) are chloroplastic enzymes, while glucan, water dikinase 2 (GWD2) is cytosolic. Both GWDs and PWD catalyze the addition of phosphate groups to amylopectin chains at the surface of starch granules, changing its physicochemical properties. As a result, GWD1 and PWD have a positive effect on transitory starch degradation at night. Because of its cytosolic localization, GWD2 does not have the same effect. Single T-DNA mutants of either GWD1 or PWD or GWD2 have been analyzed during the entire life cycle of A. thaliana. We report that the three dikinases are all important for proper seed development. Seeds from gwd2 mutants are shrunken, with the epidermal cells of the seed coat irregularly shaped. Moreover, gwd2 seeds contain a lower lipid to protein ratio and are impaired in germination. Similar seed phenotypes were observed in pwd and gwd1 mutants, except for the normal morphology of epidermal cells in gwd1 seed coats. The gwd1, pwd and gwd2 mutants were also very similar in growth and flowering time when grown under continuous light and all three behaved differently from wild-type plants. Besides pinpointing a novel role of GWD2 and PWD in seed development, this analysis suggests that the phenotypic features of the dikinase mutants in A. thaliana cannot be explained solely in terms of defects in leaf starch degradation at night.