Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.

Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.

BACKGROUND: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. METHODS: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. RESULTS: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. CONCLUSIONS: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.

Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom Thalassiosira pseudonana Using a Multiprong Approach.

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3-. The "iota" class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium-calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat "drone-like" shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.

External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm.

The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3-) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3- use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3- use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3- use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3- transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3- to CO2 at the plasmalemma by α-CA1 and transport of HCO3- across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems.

Structural basis for C4 photosynthesis without Kranz anatomy in leaves of the submerged freshwater plant Ottelia alismoides.

BACKGROUND AND AIMS: Ottelia alismoides (Hydrocharitaceae) is a freshwater macrophyte that, unusually, possesses three different CO2-concentrating mechanisms. Here we describe its leaf anatomy and chloroplast ultrastructure, how these are altered by CO2 concentration and how they may underlie C4 photosynthesis. METHODS: Light and transmission electron microscopy were used to study the anatomy of mature leaves of O. alismoides grown at high and low CO2 concentrations. Diel acid change and the activity of phosphoenolpyruvate carboxylase were measured to confirm that CAM activity and C4 photosynthesis were present. KEY RESULTS: When O. alismoides was grown at low CO2, the leaves performed both C4 and CAM photosynthesis whereas at high CO2 leaves used C4 photosynthesis. The leaf comprised an upper and lower layer of epidermal cells separated by a large air space occupying about 22 % of the leaf transverse-section area, and by mesophyll cells connecting the two epidermal layers. Kranz anatomy was absent. At low CO2, chloroplasts in the mesophyll cells were filled with starch even at the start of the photoperiod, while epidermal chloroplasts contained small starch grains. The number of chloroplasts in the epidermis was greater than in the mesophyll cells. At high CO2, the structure was unchanged but the thicknesses of the two epidermal layers, the air space, mesophyll and the transverse-section area of cells and air space were greater. CONCLUSIONS: Leaves of O. alismoides have epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy. The high starch content of mesophyll cells suggests they may benefit from an internal source of CO2, for example via C4 metabolism, and are also sites of starch storage. The air spaces may help in the recycling of decarboxylated or respired CO2. The structural similarity of leaves at low and high CO2 is consistent with the constitutive nature of bicarbonate and C4 photosynthesis. There is sufficient structural diversity within the leaf of O. alismoides to support dual-cell C4 photosynthesis even though Kranz anatomy is absent.

Insights on the Functions and Ecophysiological Relevance of the Diverse Carbonic Anhydrases in Microalgae.

Carbonic anhydrases (CAs) exist in all kingdoms of life. They are metalloenzymes, often containing zinc, that catalyze the interconversion of bicarbonate and carbon dioxide-a ubiquitous reaction involved in a variety of cellular processes. So far, eight classes of apparently evolutionary unrelated CAs that are present in a large diversity of living organisms have been described. In this review, we focus on the diversity of CAs and their roles in photosynthetic microalgae. We describe their essential role in carbon dioxide-concentrating mechanisms and photosynthesis, their regulation, as well as their less studied roles in non-photosynthetic processes. We also discuss the presence in some microalgae, especially diatoms, of cambialistic CAs (i.e., CAs that can replace Zn by Co, Cd, or Fe) and, more recently, a CA that uses Mn as a metal cofactor, with potential ecological relevance in aquatic environments where trace metal concentrations are low. There has been a recent explosion of knowledge about this well-known enzyme with exciting future opportunities to answer outstanding questions using a range of different approaches.

Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of Thiol Groups.

Widely used silver nanoparticles (AgNPs) are readily accessible to biological fluids and then surrounded by proteins. However, interactions between AgNPs and proteins are poorly understood. Two dehydrogenases, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase (MDH), are chosen to investigate these interactions. Ag bound to thiol groups of these enzymes significantly decreases the number of free thiols available. Dose-dependent inhibition of enzyme activities is observed in both AgNPs and Ag+ treatments. Based on the concentration required to inhibit 50% activity, GAPDH and MDH are 24-30 fold more sensitive to Ag+ than to AgNPs suggesting that the measured 4.2% Ag+ containing AgNPs can be responsible for the enzymes inhibition. GAPDH, with a thiol group in its active site, is more sensitive to Ag than MDH, displaying many thiol groups but none in its active site, suggesting that thiol groups at the active site strongly determines the sensitivity of enzymes toward AgNPs. In contrast, the dramatic changes of circular dichroism spectra show that the global secondary structure of MDH under AgNPs treatment is more altered than that of GAPDH. In summary, this study shows that the thiol groups and their location on these dehydrogenases are crucial for the AgNPs effects.

Orchestration of algal metabolism by protein disorder.

Intrinsically disordered proteins (IDPs) are proteins that provide many functional advantages in a large number of metabolic and signalling pathways. Because of their high flexibility that endows them with pressure-, heat- and acid-resistance, IDPs are valuable metabolic regulators that help algae to cope with extreme conditions of pH, temperature, pressure and light. They have, however, been overlooked in these organisms. In this review, we present some well-known algal IDPs, including the conditionally disordered CP12, a protein involved in the regulation of CO2 assimilation, as probably the best known example, whose disorder content is strongly dependent on the redox conditions, and the essential pyrenoid component 1 that serves as a scaffold for ribulose-1, 5-bisphosphate carboxylase/oxygenase. We also describe how some enzymes are regulated by protein regions, called intrinsically disordered regions (IDRs), such as ribulose-1, 5-bisphosphate carboxylase/oxygenase activase, the A2B2 form of glyceraldehyde-3-phosphate dehydrogenase and the adenylate kinase. Several molecular chaperones, which are crucial for cell proteostasis, also display significant disorder propensities such as the algal heat shock proteins HSP33, HSP70 and HSP90. This review confirms the wide distribution of IDPs in algae but highlights that further studies are needed to uncover their full role in orchestrating algal metabolism.

Exploring the microbiome of the "star" freshwater diatom Asterionella formosa in a laboratory context.

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies.

Different CO2 acclimation strategies in juvenile and mature leaves of Ottelia alismoides.

The freshwater macrophyte, Ottelia alismoides, is a bicarbonate user performing C4 photosynthesis in the light, and crassulacean acid metabolism (CAM) when acclimated to low CO2. The regulation of the three mechanisms by CO2 concentration was studied in juvenile and mature leaves. For mature leaves, the ratios of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) are in the range of that of C4 plants regardless of CO2 concentration (1.5-2.5 at low CO2, 1.8-3.4 at high CO2). In contrast, results for juvenile leaves suggest that C4 is facultative and only present under low CO2. pH-drift experiments showed that both juvenile and mature leaves can use bicarbonate irrespective of CO2 concentration, but mature leaves have a significantly greater carbon-extracting ability than juvenile leaves at low CO2. At high CO2, neither juvenile nor mature leaves perform CAM as indicated by lack of diurnal acid fluctuation. However, CAM was present at low CO2, though the fluctuation of titratable acidity in juvenile leaves (15-17 µequiv g-1 FW) was slightly but significantly lower than in mature leaves (19-25 µequiv g-1 FW), implying that the capacity to perform CAM increases as leaves mature. The increased CAM activity is associated with elevated PEPC activity and large diel changes in starch content. These results show that in O. alismoides, carbon-dioxide concentrating mechanisms are more effective in mature compared to juvenile leaves, and C4 is facultative in juvenile leaves but constitutive in mature leaves.

Ecological imperatives for aquatic CO2-concentrating mechanisms.

In aquatic environments, the concentration of inorganic carbon is spatially and temporally variable and CO2 can be substantially oversaturated or depleted. Depletion of CO2 plus low rates of diffusion cause inorganic carbon to be more limiting in aquatic than terrestrial environments, and the frequency of species with a CO2-concentrating mechanism (CCM), and their contribution to productivity, is correspondingly greater. Aquatic photoautotrophs may have biochemical or biophysical CCMs and exploit CO2 from the sediment or the atmosphere. Though partly constrained by phylogeny, CCM activity is related to environmental conditions. CCMs are absent or down-regulated when their increased energy costs, lower CO2 affinity, or altered mineral requirements outweigh their benefits. Aquatic CCMs are most widespread in environments with low CO2, high HCO3-, high pH, and high light. Freshwater species are generally less effective at inorganic carbon removal than marine species, but have a greater range of ability to remove carbon, matching the environmental variability in carbon availability. The diversity of CCMs in seagrasses and marine phytoplankton, and detailed mechanistic studies on larger aquatic photoautotrophs are understudied. Strengthening the links between ecology and CCMs will increase our understanding of the mechanisms underlying ecological success and will place mechanistic studies in a clearer ecological context.

Diversity of CO2-concentrating mechanisms and responses to CO2 concentration in marine and freshwater diatoms.

The presence of CO2-concentrating mechanisms (CCMs) is believed to be one of the characteristics that allows diatoms to thrive in many environments and to be major contributors to global productivity. Here, the type of CCM and the responses to variable CO2 concentration were studied in marine and freshwater diatoms. At 400 ppm, there was a large diversity in physiological and biochemical mechanisms among the species. While Phaeodactylum tricornutum mainly used HCO3-, Thalassiosira pseudonana mainly used CO2. Carbonic anhydrase was an important component of the CCM in all species and C4 metabolism was absent, even with T. weissflogii. For all species, at 20 000 ppm, the affinity for dissolved inorganic carbon was lower than at 400 ppm CO2 and the reliance on CO2 was higher. Despite the difference in availability of inorganic carbon in marine and fresh waters, there were only small differences in CCMs between species from the two environments, and Navicula pelliculosa behaved similarly when grown in the two environments. The results suggest that species-specific differences are great, and more important than environmental differences in determining the nature and effectiveness of the CCM in diatoms.

Responses of Ottelia alismoides, an aquatic plant with three CCMs, to variable CO2 and light.

Ottelia alismoides is a constitutive C4 plant and bicarbonate user, and has facultative crassulacean acid metabolism (CAM) at low CO2. Acclimation to a factorial combination of light and CO2 showed that the ratio of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (>5) is in the range of that of C4 plants. This and short-term response experiments showed that the activity of PEPC and pyruvate phosphate dikinase (PPDK) was high even at the end of the night, consistent with night-time acid accumulation and daytime carbon fixation. The diel acidity change was maximal at high light and low CO2 at 17-25 µequiv g-1 FW. Decarboxylation proceeded at ~2-3 µequiv g-1 FW h-1, starting at the beginning of the photoperiod, but did not occur at high CO2; the rate was greater at high, compared with low light. There was an inverse relationship between starch formation and acidity loss. Acidity changes account for up to 21% of starch production and stimulate early morning photosynthesis, but night-time accumulation of acid traps <6% of respiratory carbon release. Ottelia alismoides is the only known species to operate CAM and C4 in the same tissue, and one of only two known aquatic species to operate CAM and bicarbonate use.

Redox regulation of ATP sulfurylase in microalgae.

ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS.

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state.

The redox switch protein CP12 is a key player of the regulation of the Benson-Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold.

The nature of the CO2 -concentrating mechanisms in a marine diatom, Thalassiosira pseudonana.

Diatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide-concentrating mechanisms (CCMs) involving transporters and carbonic anhydrases (CAs) are well known, but the contribution of a biochemical CCM involving C4 metabolism is contentious. The CCM(s) present in the marine-centric diatom, Thalassiosira pseudonana, were studied in cells exposed to high or low concentrations of CO2 , using a range of approaches. At low CO2 , cells possessed a CCM based on active uptake of CO2 (70% contribution) and bicarbonate, while at high CO2 , cells were restricted to CO2 . CA was highly and rapidly activated on transfer to low CO2 and played a key role because inhibition of external CA produced uptake kinetics similar to cells grown at high CO2 . The activities of phosphoenolpyruvate (PEP) carboxylase (PEPC) and the PEP-regenerating enzyme, pyruvate phosphate dikinase (PPDK), were lower in cells grown at low than at high CO2 . The ratios of PEPC and PPDK to ribulose bisphosphate carboxylase were substantially lower than 1, even at low CO2 . Our data suggest that the kinetic properties of this species results from a biophysical CCM and not from C4 type metabolism.

Micellar lipid composition affects micelle interaction with class B scavenger receptor extracellular loops.

Scavenger receptors (SRs) like cluster determinant 36 (CD36) and SR class B type I (SR-BI) play a debated role in lipid transport across the intestinal brush border membrane. We used surface plasmon resonance to analyze real-time interactions between the extracellular protein loops and various ligands ranging from single lipid molecules to mixed micelles. Micelles mimicking physiological structures were necessary for optimal binding to both the extracellular loop of CD36 (lCD36) and the extracellular loop of SR-BI (lSR-BI). Cholesterol, phospholipid, and fatty acid micellar content significantly modulated micelle binding to and dissociation from the transporters. In particular, high phospholipid micellar concentrations inhibited micelle binding to both receptors (-53.8 and -74.4% binding at 0.32 mM compared with 0.04 mM for lCD36 and lSR-BI, respectively, P < 0.05). The presence of fatty acids was crucial for micelle interactions with both proteins (94.4 and 81.3% binding with oleic acid for lCD36 and lSR-BI, respectively, P < 0.05) and fatty acid type substitution within the micelles was the component that most impacted micelle binding to the transporters. These effects were partly due to subsequent modifications in micellar size and surface electric charge, and could be correlated to micellar vitamin D uptake by Caco-2 cells. Our findings show for the first time that micellar lipid composition and micellar properties are key factors governing micelle interactions with SRs.

The DJ-1 superfamily member Hsp31 repairs proteins from glycation by methylglyoxal and glyoxal.

Hsp31 belongs to the PfpI/Hsp31/DJ-1 superfamily, and has been reported to display chaperone, peptidase and glutathione-independent glyoxalase activities. Here, we show that Hsp31 repairs glyoxal- and methylglyoxal-glycated amino acids and proteins and releases repaired proteins and lactate or glycolate, respectively. Hsp31 deglycates cysteine, arginine and lysine by acting on early glycation intermediates (hemithioacetals and aminocarbinols) and prevents the formation of Schiff bases and advanced glycation endproducts. Hsp31 repairs glycated serum albumin, glyceraldehyde-3-phosphate dehydrogenase, fructose biphosphate aldolase and aspartate aminotransferase. Moreover, we show that bacterial extracts from the hchA mutant display increased glycation levels and that the apparent glyoxalase activity of Hsp31 reflects its deglycase activity. Our results suggest that other Hsp31 members, previously characterized as glutathione-independent glyoxalases, likely function as protein deglycases.

FRET analysis of CP12 structural interplay by GAPDH and PRK.

CP12 is an intrinsically disordered protein playing a key role in the regulation of the Benson-Calvin cycle. Due to the high intrinsic flexibility of CP12, it is essential to consider its structural modulation induced upon binding to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) enzymes. Here, we report for the first time detailed structural modulation about the wild-type CP12 and its site-specific N-terminal and C-terminal disulfide bridge mutants upon interaction with GAPDH and PRK by Förster resonance energy transfer (FRET). Our results indicate an increase in CP12 compactness when the complex is formed with GAPDH or PRK. In addition, the distributions in FRET histograms show the elasticity and conformational flexibility of CP12 in all supra molecular complexes. Contrarily to previous beliefs, our FRET results importantly reveal that both N-terminal and C-terminal site-specific CP12 mutants are able to form the monomeric (GAPDH-CP12-PRK) complex.