Ricardo A. Wolosiuk

Thioredoxin and enzyme regulation

Abstract Thioredoxin, a hydrogen carrier protein that functions in DNA synthesis and in the transformation of sulfur metabolites, has recently been found to serve as a regulatory protein in linking light to the activation of enzymes during photosynthesis.

The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond

Experiments initiated in the early 1960s on fermentative bacteria led to the discovery of ferredoxin-dependent alpha-ketocarboxylation reactions that were later found to be key to a new cycle for the assimilation of carbon dioxide in photosynthetic bacteria (the reductive carboxylic acid or reverse citric cycle). The latter finding set the stage for the discovery of a regulatory system, the ferredoxin/thioredoxin system, functional in photosynthesis in chloroplasts and oxygen-evolving photosynthetic prokaryotes. The chloroplast research led to a description of the extraplastidic NADP/thioredoxin system that is now known to function in heterotrophic plant processes such as seed germination and self-incompatibility. Extensions of the fundamental research have begun to open doors to the broad application of thioredoxin in technology and medicine.

Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system.

Abstract A newly found form of chloroplast phosphoribulokinase (designated the “regulatory form”) required reduced thioredoxin for activity. A second form of the enzyme (the “nonregulatory form”) was not appreciably affected by thioredoxin. The thioredoxin required for activation of the regulatory enzyme could be reduced (i) photochemically by chloroplast membranes that were supplemented with ferredoxin and ferredoxin-thioredoxin reductase or (ii) chemically in the dark with the sulfhydryl reagent dithiothreitol. Following activation by reduced thioredoxin, phosphoribulokinase was deactivated by the soluble chloroplast oxidants dehydroascorbate and oxidized glutathione. The results suggest that the regulatory form of phosphoribulokinase resembles fructose 1,6-bisphosphatase in its mode of regulation by the ferredoxin/thioredoxin system.

Regulation of NADP‐malate dehydrogenase by the light‐actuated ferredoxin/thioredoxin system of chloroplasts

We have recently described a new regulatory system of chloroplasts whereby enzymes are activated in the light by reduction and are deactivated in the dark by oxidation [ 11. Activation is achieved in a soluble system that consists of: (i) Ferredoxin, the strongly electronegative acceptor of photosynthetic electron transport. (ii) Thioredoxin, a hydrogen carrier protein that is reduced photochemically via ferredoxin. (iii) Ferredoxin-thiotedoxin reductase, a newly-found enzyme that catalyzes the reduction of thioredoxin by photoreduced ferredoxin (eq. l-3).

Two proteins function in the regulation of photosynthetic CO2 assimilation in chloroplasts

THERE is increasing evidence that products of the light reactions of photosynthesis govern the activity of enzymes involved in CO2 assimilation by chloroplasts1–15. Of these products, reductants formed photochemically seem to be of particular importance. Such reductants include the reduced form of ferredoxin2,6,10, a strongly electronegative chloroplast iron-sulphur protein (E′0=−0.42 V) that activates the two key chloroplast enzymes fructose 1,6-bis-phosphatase and sedoheptulose l,7-bis-phosphatase. Activation of both of these enzymes requires in addition to reduced ferredoxin a ‘protein factor’ that is indigenous to chloroplasts. In efforts to elucidate the nature of the ferredoxin-linked enzyme activation, we have separated the protein factor into two components16: (1) a partly purified protein, provisionally named “assimilation regulatory protein a” (ARPa) and (2) a highly purified chromophore-free protein called “assimilation regulatory protein b” (ARPb). Only the latter was required for activation when reduced ferredoxin was replaced by the non-physiological sulphydryl reagent dithiothreitol6,10.

Typical 2-Cys peroxiredoxins – modulation by covalent transformations and noncovalent interactions

2‐Cys peroxiredoxins are peroxidases devoid of prosthetic groups that mediate in the defence against oxidative stress and the peroxide activation of signaling pathways. This dual capacity relies on the high reactivity of the conserved peroxidatic and resolving cysteines, whose modification embraces not only the usual thiol–disulfide exchange but also higher oxidation states of the sulfur atom. These changes are part of a complex system wherein the cooperation with other post‐translational modifications – phosphorylation, acetylation – may function as major regulatory mechanisms of the quaternary structure. More importantly, modern proteomic approaches have identified the oxyacids at cysteine residues as novel protein targets for unsuspected post‐translational modifications, such as phosphorylation that yields the unusual sulfi(o)nic–phosphoryl anhydride. In this article, we review the biochemical attributes of 2‐Cys peroxiredoxins that, in combination with complementary studies of forward and reverse genetics, have generated stimulating molecular models to explain how this enzyme integrates into cell signaling in vivo.

A dual effect of Ca2+ on chloroplast fructose-1,6-bisphosphatase

Summary Chloroplast fructose-1,6-bisphosphatase, isolated from spinach leaves, was activated by preincubation with Ca2+ (or Mn2+), fructose-1,6-bisphosphate and dithiothreitol-reduced thioredoxin-f. Upon activation, the enzyme displayed high activity when measured at low concentrations of both fructose-1,6-bisphosphate and Mg2+. On the contrary, the activity of chloroplast fructose-1,6-bisphosphatase was inhibited by Ca2+. These results suggest that Ca2+ (or Mn2+) is potentially important in the regulation of the chloroplast fructose-1,6-bisphosphatase reaction (activation and catalysis).

Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants.

Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f.

Appearance of sedoheptulose 1,7-diphosphatase activity on conversion of chloroplast fructose 1,6-diphosphatase from dimer form to monomer form.

Chloroplast fructose 1,6-diphosphatase isolated at pH 5.5 as the dimer dissociated to the monomer at pH 8.5. When the pH was adjusted from 8.5 back to 5.5, the newly formed monomer partly reassociated to form the dimer. The monomer lacked the fructose diphosphatase activity characteristic of the dimer (measured in the presence of a saturating concentration of Mg++) but retained ferredoxin-dependent activity (measured in the presence of Mg++ plus protein factor and either reduced ferredoxin or dithiothreitol). In addition, the monomer acquired sedoheptulose 1,7-diphosphatase activity that was dependent on either reduced ferredoxin or dithiothreitol and the protein factor.

Role of electrostatic interactions on the affinity of thioredoxin for target proteins. Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1,6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. colithioredoxin for chloroplast fructose-1,6-bisphosphatase (A 0.5 = 9, 7, and 3 μm, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A 0.5 = 33 μm), though still lower than that of thioredoxin-f (A 0.5 = 0.9 μm). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A 0.5for all bacterial variants to a limiting value of ∼9 μmbut also increases the A 0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.