Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation.

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.     

Molecular mechanism of NADPH-glyceraldehyde-3-phosphate dehydrogenase regulation through the C-terminus of CP12 in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.     

SPECIFICITY AND FUNCTION OF GLYCERALDEHYDE‐3‐PHOSPHATE DEHYDROGENASE IN A FRESHWATER DIATOM,ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1

The plastidic glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) catalyzes the only reductive step in the Calvin cycle and exists as different forms of which GapC1 enzyme is present in chromalveolates, such as diatoms. Biochemical studies on diatoms are still fragmentary, and, thus, in this report, GAPDH from the freshwater diatom Asterionella formosa Hassall has been purified and kinetically characterized. It is a homotetrameric enzyme with a molecular mass of ~150 ± 15 kDa. The enzyme showed Michaelis–Menten kinetics with respect to both cofactors, NADPH and NADH, with a 16‐fold greater catalytic constant for NADPH. The K_m for NADPH was 140 μM, the lowest affinity reported, while the catalytic constant, 815 s^?1, is the highest reported. The K_m for NADH was 93 μM, and the catalytic constant was 50 s^?1, both are similar to reported values for other types of GAPDH. The GapC1 enzyme, like the Chlamydomonas reinhardtii A₄ GAPDH, exhibits a cooperative behavior toward the substrate, 1,3‐bisphosphoglyceric acid (BPGA), with both cofactors. Mass spectrometry analysis showed that when GapC1 enzyme was purified without reducing agents, it copurified with a small protein with a mass of 8.2 kDa. This protein was recognized by antibodies against CP12. When associated with this protein, GAPDH displayed a lag that disappeared upon incubation with reducing agent in the presence of either BPGA or NADPH as a consequence of dissociation of the GAPDH/CP12 complex. Thus, as in other species of algae and higher plants, regulation of GapC1 enzyme in A. formosa may occur through association‐dissociation processes linked to dark‐light transitions.     

Reconstitution and properties of the recombinant glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase supramolecular complex of Arabidopsis

Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the regulatory peptide CP12 a supramolecular complex in Arabidopsis (Arabidopsis thaliana) that could be reconstituted in vitro using purified recombinant proteins. Both enzyme activities were strongly influenced by complex formation, providing an effective means for regulation of the Calvin cycle in vivo. PRK and CP12, but not GapA (A(4) isoform of GAPDH), are redox-sensitive proteins. PRK was reversibly inhibited by oxidation. CP12 has no enzymatic activity, but it changed conformation depending on redox conditions. GapA, a bispecific NAD(P)-dependent dehydrogenase, specifically formed a binary complex with oxidized CP12 when bound to NAD. PRK did not interact with either GapA or CP12 singly, but oxidized PRK could form with GapA/CP12 a stable ternary complex of about 640 kD (GapA/CP12/PRK). Exchanging NADP for NAD, reducing CP12, or reducing PRK were all conditions that prevented formation of the complex. Although GapA activity was little affected by CP12 alone, the NADPH-dependent activity of GapA embedded in the GapA/CP12/PRK complex was 80% inhibited in respect to the free enzyme. The NADH activity was unaffected. Upon binding to GapA/CP12, the activity of oxidized PRK dropped from 25% down to 2% the activity of the free reduced enzyme. The supramolecular complex was dissociated by reduced thioredoxins, NADP, 1,3-bisphosphoglycerate (BPGA), or ATP. The activity of GapA was only partially recovered after complex dissociation by thioredoxins, NADP, or ATP, and full GapA activation required BPGA. NADP, ATP, or BPGA partially activated PRK, but full recovery of PRK activity required thioredoxins. The reversible formation of the GapA/CP12/PRK supramolecular complex provides novel possibilities to finely regulate GapA ("non-regulatory" GAPDH isozyme) and PRK (thioredoxin sensitive) in a coordinated manner.     

Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH/CP12 complex. Consequently, a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central alpha-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH/CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5-75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcal.mol(-1) destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.     

Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.     

NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst.

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.     

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.     

Dual coenzyme specificity of photosynthetic glyceraldehyde-3-phosphate dehydrogenase interpreted by the crystal structure of A4 isoform complexed with NAD

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms.     

Regulation of phosphoribulokinase and glyceraldehyde 3‐phosphate dehydrogenase in a freshwater diatom,Asterionella formosa1

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.     

Spontaneous assembly of photosynthetic supramolecular complexes as mediated by the intrinsically unstructured protein CP12

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-GAPDH)-(CP12-2)(2) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-GAPDH)-(CP12-2)(2)-(PRK))(2). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAPDH and PRK during darkness in photosynthetic tissues.     

Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis.The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes.Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.     

The small protein CP12: a protein linker for supramolecular complex assembly

CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants. It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12. The role of CP12 in this complex assembly has not been determined. To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli. Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12. On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). This unit probably dimerizes due partially to interactions between the enzymes of each unit. Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions. Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured. Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly. CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants.     

Antisense suppression of the small chloroplast protein CP12 in tobacco alters carbon partitioning and severely restricts growth

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.