Sandra Scagliarini

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.

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m−2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m−2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher “dark” activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.

Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate

Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP+ and dithiothreitol consisted of highly NADPH-active protomers of 160 kDa; otherwise, it always consisted of a 600-kDa oligomer (regulatory form) favoured by the addition of NAD+ in buffers and with low NADPH-dependent activity (ratio of activities with NADPH versus NADH of 0.2–0.4). Glycerate 1,3-bisphosphate (BPGA) was prepared enzymatically using rabbit-muscle NAD-GAPDH, and purified. Among known modulators of spinach NAD(P)-GAPDH, BPGA is the most effective on a molar basis in stimulating NADPH-activity of “dark” chloroplast extracts and purified NAD(P)-GAPDH (activation constant, Ka= 12 μM). It also causes the enzyme to dissociate into 160-kDa protomers. The Km of BPGA both with NADPH or NADH as coenzyme is 4–7 μM. NAD+ and NADH are inhibitory to the activation process induced by BPGA. This compound, together with NADP(H) and ATP belongs to a group of substrate-modifiers of the NADPH-activity and conformational state of spinach NAD(P)-GAPDH, all characterized by Ka values three- to tenfold higher than the Km. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts Heineke et al. 1991, Plant Physiol. 95, 1131–1137, it is proposed that NAD+ promotes NAD(P)-GAPDH association into a regulatory conformer with low NADPH-activity during dark deactivation. The process is reversed in the light by BPGA and other substrate-modifiers whose concentration increases during photosynthesis, in addition to reduced thioredoxin.

NADH : Fe (III) -chelate reductase of maize roots is an active cytochrome b5 reductase

Microsomal NADH:Fe(III)‐chelate reductase (NFR) of maize roots has been purified as a monomeric flavoprotein of 32 kDa with non‐covalently bound FAD. In the presence of NADH, NFR efficiently reduced the physiological iron‐chelate Fe(III)‐citrate (K cat/K m(Fe(III)‐citrate)=6.0×106 M−1 s−1) with a sequential reaction mechanism. Purified NFR was totally inhibited by the sulfhydryl reagent PHMB at 10−9 M, and it could use cyt b 5 as alternative electron acceptor with a maximal reduction rate as high as with Fe(III)‐citrate. We conclude that in maize roots the reduction of Fe(III)‐citrate is chiefly performed by a cytochrome b 5 reductase, mostly associated with intracellular membranes and in part with the plasma membrane.

Arguments against a close relationship between non-phosphorylating and phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Non‐phosphorylating NADP‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (EC 1.2.1.9) from spinach leaves was purified to homogeneity using an improved purification procedure. Thus, a major contaminant with molecular mass and ion‐exchange properties similar to non‐phosphorylating GAPDH was eliminated. Using this pure non‐phosphorylating GAPDH, cofactor stereospecificity was determined by 1H NMR. Analysis of the NADPH formed from the hydride transfer from glyceraldehyde‐3‐phosphate to [4‐2H]NADP showed that the enzyme belongs to the A‐stereospecific dehydrogenase family. This stereospecificity is the same as that described for the aldehyde dehydrogenase (ALDH) superfamily and opposite to that of the phosphorylating GAPDH. Moreover, results from peptide sequencing analysis suggest a similarity in sequence between the non‐phosphorylating GAPDH and ALDHs. Thus, the results taken all together strongly suggest that non‐phosphorylating GAPDH belongs to the ALDH family and has no close relationship to the phosphorylating GAPDH class.

Crystallization and preliminary X-ray study of chloroplast glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts has been crystallized by vapour diffusion in the pH range 7-8.5 in (NH4)2SO4 and Tris-HCl buffer or potassium phosphate buffer at room temperature. Crystals of the A4 isoform, grown at pH 8.5 in Tris-HCl buffer, diffract to 3.0 A (at 100 K) using synchrotron radiation. The crystals belong to the orthorhombic C222 space group, with unit-cell dimensions a = 145.9, b = 185.9 and c = 106.3 A, and probably contain one tetramer per asymmetric unit. Structure determination by molecular replacement is in progress.