Characterization of chloroplast ribulose-5-phosphate-3-epimerase from the microalga Chlamydomonas reinhardtii.

Carbon fixation relies on Rubisco and 10 additional enzymes in the Calvin-Benson-Bassham cycle. Epimerization of xylulose-5-phosphate (Xu5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco. Ribulose-5-phosphate-3-epimerase (RPE, EC 5.1.3.1) catalyzes the formation of Ru5P, but it can also operate in the pentose-phosphate pathway by catalyzing the reverse reaction. Here, we describe the structural and biochemical properties of the recombinant RPE isoform 1 from Chlamydomonas (Chlamydomonas reinhardtii) (CrRPE1). The enzyme is a homo-hexamer that contains a zinc ion in the active site and exposes a catalytic pocket on the top of an α8ß8 triose isomerase-type barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. By optimizing and developing enzyme assays to monitor the reversible epimerization of Ru5P to Xu5P and vice versa, we determined the catalytic parameters that differ from those of other plant paralogs. Despite being identified as a putative target of multiple thiol-based redox modifications, CrRPE1 activity is not affected by both reductive and oxidative treatments, indicating that enzyme catalysis is insensitive to possible redox alterations of cysteine residues. 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. This work provides an accurate description of the structural features of CrRPE1 and an in-depth examination of its catalytic and regulatory properties highlighting the physiological relevance of this enzyme in the context of photosynthetic carbon fixation.

Dark complexes of the Calvin-Benson cycle in a physiological perspective.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two enzymes of the Calvin Benson cycle that stand out for some peculiar properties they have in common: (i) they both use the products of light reactions for catalysis (NADPH for GAPDH, ATP for PRK), (ii) they are both light-regulated through thioredoxins and (iii) they are both involved in the formation of regulatory supramolecular complexes in the dark or low photosynthetic conditions, with or without the regulatory protein CP12. In the complexes, enzymes are transiently inactivated but ready to recover full activity after complex dissociation. Fully active GAPDH and PRK are in large excess for the functioning of the Calvin-Benson cycle, but they can limit the cycle upon complex formation. Complex dissociation contributes to photosynthetic induction. CP12 also controls PRK concentration in model photosynthetic organisms like Arabidopsis thaliana and Chlamydomonas reinhardtii. The review combines in vivo and in vitro data into an integrated physiological view of the role of GAPDH and PRK dark complexes in the regulation of photosynthesis.

Proline, Cysteine and Branched-Chain Amino Acids in Abiotic Stress Response of Land Plants and Microalgae.

Proteinogenic amino acids are the building blocks of protein, and plants synthesize all of them. In addition to their importance in plant growth and development, growing evidence underlines the central role played by amino acids and their derivatives in regulating several pathways involved in biotic and abiotic stress responses. In the present review, we illustrate (i) the role of amino acids as an energy source capable of replacing sugars as electron donors to the mitochondrial electron transport chain and (ii) the role of amino acids as precursors of osmolytes as well as (iii) precursors of secondary metabolites. Among the amino acids involved in drought stress response, proline and cysteine play a special role. Besides the large proline accumulation occurring in response to drought stress, proline can export reducing equivalents to sink tissues and organs, and the production of H2S deriving from the metabolism of cysteine can mediate post-translational modifications that target protein cysteines themselves. Although our general understanding of microalgae stress physiology is still fragmentary, a general overview of how unicellular photosynthetic organisms deal with salt stress is also provided because of the growing interest in microalgae in applied sciences.

Conformational Disorder Analysis of the Conditionally Disordered Protein CP12 from Arabidopsis thaliana in Its Different Redox States.

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.

Arabidopsis thaliana Sucrose Phosphate Synthase A2 Affects Carbon Partitioning and Drought Response.

Sucrose is essential for plants for several reasons: It is a source of energy, a signaling molecule, and a source of carbon skeletons. Sucrose phosphate synthase (SPS) catalyzes the conversion of uridine diphosphate glucose and fructose-6-phosphate to sucrose-6-phosphate, which is rapidly dephosphorylated by sucrose phosphatase. SPS is critical in the accumulation of sucrose because it catalyzes an irreversible reaction. In Arabidopsis thaliana, SPSs form a gene family of four members, whose specific functions are not clear yet. In the present work, the role of SPSA2 was investigated in Arabidopsis under both control and drought stress conditions. In seeds and seedlings, major phenotypic traits were not different in wild-type compared with spsa2 knockout plants. By contrast, 35-day-old plants showed some differences in metabolites and enzyme activities even under control conditions. In response to drought, SPSA2 was transcriptionally activated, and the divergences between the two genotypes were higher, with spsa2 showing reduced proline accumulation and increased lipid peroxidation. Total soluble sugars and fructose concentrations were about halved compared with wild-type plants, and the plastid component of the oxidative pentose phosphate pathway was activated. Unlike previous reports, our results support the involvement of SPSA2 in both carbon partitioning and drought response.

Ribulose-1,5-bisphosphate regeneration in the Calvin-Benson-Bassham cycle: Focus on the last three enzymatic steps that allow the formation of Rubisco substrate.

The Calvin-Benson-Bassham (CBB) cycle comprises the metabolic phase of photosynthesis and is responsible for carbon fixation and the production of sugar phosphates. The first step of the cycle involves the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which catalyzes the incorporation of inorganic carbon into 3-phosphoglyceric acid (3PGA). The following steps include ten enzymes that catalyze the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco. While it is well established that Rubisco activity acts as a limiting step of the cycle, recent modeling studies and experimental evidence have shown that the efficiency of the pathway is also impacted by the regeneration of the Rubisco substrate itself. In this work, we review the current understanding of the structural and catalytic features of the photosynthetic enzymes that catalyze the last three steps of the regeneration phase, namely ribose-5-phosphate isomerase (RPI), ribulose-5-phosphate epimerase (RPE), and phosphoribulokinase (PRK). In addition, the redox- and metabolic-based regulatory mechanisms targeting the three enzymes are also discussed. Overall, this review highlights the importance of understudied steps in the CBB cycle and provides direction for future research aimed at improving plant productivity.

Unravelling the regulation pathway of photosynthetic AB-GAPDH.

Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC-SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.

A dominant mutation in ß-AMYLASE1 disrupts nighttime control of starch degradation in Arabidopsis leaves.

Arabidopsis (Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 (BAM1; mutant allele named bam1-2D), resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.

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

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.

Calvin-Benson cycle regulation is getting complex.

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.

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii.

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.

Impact of Drought on Soluble Sugars and Free Proline Content in Selected Arabidopsis Mutants.

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 δ1-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.

The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation.

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pK a values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pK a1 = 5.70 ± 0.28; pK a2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed.

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

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 ß-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.

Structural and Biochemical Insights into the Reactivity of Thioredoxin h1 from Chlamydomonas reinhardtii.

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.

Redox Regulation of Starch Metabolism.

Metabolism of starch is a major biological integrator of plant growth supporting nocturnal energy dynamics by transitory starch degradation as well as periods of dormancy, re-growth, and reproduction by utilization of storage starch. Especially, the extraordinarily well-tuned and coordinated rate of transient starch biosynthesis and degradation suggests the presence of very sophisticated regulatory mechanisms. Together with the circadian clock, land plants (being autotrophic and sessile organisms) need to monitor, sense, and recognize the photosynthetic rate, soil mineral availability as well as various abiotic and biotic stress factors. Currently it is widely accepted that post-translational modifications are the main way by which the diel periodic activity of enzymes of transient starch metabolism are regulated. Among these mechanisms, thiol-based redox regulation is suggested to be of fundamental importance and in chloroplasts, thioredoxins (Trx) are tightly linked up to photosynthesis and mediate light/dark regulation of metabolism. Also, light independent NADP-thioredoxin reductase C (NTRC) plays a major role in reactive oxygen species scavenging. Moreover, Trx and NTRC systems are interconnected at several levels and strongly influence each other. Most enzymes involved in starch metabolism are demonstrated to be redox-sensitive in vitro. However, to what extent their redox sensitivity is physiologically relevant in synchronizing starch metabolism with photosynthesis, heterotrophic energy demands, and oxidative protection is still unclear. For example, many hydrolases are activated under reducing (light) conditions and the strict separation between light and dark metabolic pathways is now challenged by data suggesting degradation of starch during the light period.

The analysis of the different functions of starch-phosphorylating enzymes during the development of Arabidopsis thaliana plants discloses an unexpected role for the cytosolic isoform GWD2.

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.