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.     

Purification and properties of NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the green alga Chlamydomonas reinhardtii

NADP-dependent non-phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9), previously described in higher plants, has been now found to be present in eukaryotic green algae, but in neither cyanobacteria nor non-photosynthetic microorganisms. The enzyme from the unicellular green alga Chlamydomonas reinhardtii, strain 6145c, has been purified to apparent electrophoretic homogeneity. The non-phosphorylating enzyme was effectively separated from the NADP-dependent phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) dye-ligand chromatography on Reactive Red-120 agarose. The purified enzyme exhibited an optimum pH in the 8.5–9.0 range and a specific activity of approx. 8 μmol·(mg protein)⁻¹·min⁻¹. The native protein was characterized as a homotetramer with a molecular weight of 190 000, a Stokes radius of 5.2 mn, and an isoelectric point of 6.9. From kinetic studies, K_m-values of 9.8 and 51 μM were calculated for NADP and D-glyceraldehyde 3-phosphate, respectively, an absolute specificity for both substrates being observed. L-Glyceraldehyde 3-phosphate was a potent non-competitive inhibior (K_i, 48 μM). The reaction products NADPH and D-3-phosphoglycerate inhibited enzyme activity in a competitive manner with respect to NADP (K_i, 78 μM) and D-glyceraldehyde 3-phosphate (K_i, 1.2 mM), respectively. Thermal inactivation occurred above 45°C and was effectively prevented by either substrate. The presence of essential vicinal thiol groups is suggested by the inactivation produced by diamide, with D-glyceraldehyde 3-phosphate, but not NADP, behaving as a protective agent. The enzyme's possible physiological role in photosynthetic metabolism is discussed briefly.     

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Regulation of the Calvin–Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin–Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A₂B₂-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A₄-GAPDH. GapB subunits of A₂B₂-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A₂B₂-GAPDH into strongly inactivated A₈B₈-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A₄-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A₂B₂-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.     

Study of the interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA

Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed.The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806–2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH.A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3′-terminal adenosine and is greater when the 5′-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.     

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.     

Structure basis for the regulation of glyceraldehyde-3-phosphate dehydrogenase activity via the intrinsically disordered protein CP12

The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Memory and imprinting in multienzyme complexes. Evidence for information transfer from glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase under reduced state in Chlamydomonas reinhardtii.

The phosphoribulokinase, when it is in a reduced state in a bi-enzyme complex, is more active than when it is oxidized. This complex dissociates upon dilution to give a metastable reduced form of phosphoribulokinase, which differs from the stable form isolated beside the complex. The kinetic parameters of the reduced stable phosphoribulokinase and those of the complex are very similar, unlike those of the metastable form. Although the kinetic mechanism of the reduced stable form is ordered, with ribulose-5-phosphate binding first, ATP binds first to the phosphoribulokinase in the complex and to the metastable form. Therefore, phosphoribulokinase bears an imprint from glyceraldehyde-3-phosphate dehydrogenase after disruption of the complex. Dissociation of phosphoribulokinase from the complex also enhances its flexibility. The imprinting and greater flexibility result in the catalytic constant of dissociated phosphoribulokinase being 10-fold higher than that of the enzyme in the complex. Imprinting corresponds to stabilization-destabilization energies resulting from conformation changes generated by protein-protein interactions. The energy stored within the metastable phosphoribulokinase is mainly used to decrease the energy barrier to catalysis.     

Kinetic properties of a sn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii

A sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from the unicellular green alga Chlamydomonas reinhardtii 3400-fold to a specific activity of 34 mumol/mg protein per min by a simple procedure involving two chromatographic steps on affinity dyes. The pH optimum for reduction of dihydroxyacetone phosphate was 6.8 and for glycerol 3-phosphate oxidation it was 9.5. In the direction of dihydroxyacetone phosphate reduction, the enzyme showed Michaelis-Menten kinetics. The enzyme reacted specifically with NADH and dihydroxyacetone phosphate as substrates with affinity constants of 16 and 12 microM, respectively. Product inhibition as well as competitive inhibition pattern indicated a random-bi-bi reaction mechanism for sn-glycerol-3-phosphate dehydrogenase from C. reinhardtii. The effective control of dihydroxyacetone reduction catalysed via this enzyme by ATP, Pi and NAD gave evidence for a physiological role of the enzyme in plastidic glycolysis.     

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.     

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.     

The radiolysis of glyceraldehyde-3-phosphate dehydrogenase.

The yields in molecules per 100 eV for active-site and sulphydryl loss from glyceraldehyde-3-phosphate dehydrogenase have been determined in nitrous-oxide-saturated, aerated and argon-saturated solutions. Molecular hydrogen peroxide produces a sulphenic acid product, which can be repaired by post-irradiation treatment with dithiothreitol. Comparison of the yields under various conditions showed that in aerated solutions both .OH and .O2-radicals inactivated the enzyme with an efficiency of about 26 per cent. However, the efficiency of .OH in air-free solutions was less, and inactivation by .H and eaq- did not appear to be appreciable. There is a correlation between SH loss and loss of active sites.     

Immobilized hybrids of glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) immobilized on CNBr-activated Sepharose 4-B has been subjected to dissociation to obtain matrix-bound dimeric species of the enzyme. Hybridization was then performed using soluble glyceraldehyde-3-phosphate dehydrogenase isolated from rat skeletal muscle. Immobilized hybrid tetramers thus obtained were demonstrated to exhibit two distinct pH-optima of activity characteristic of the yeast and muscle enzymes, respectively. The results indicate that under appropriate conditions the activity of each of the dimers composing the immobilized hybrid tetramer can be studied separately.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.