Nancy A. Crawford

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).

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Enzyme regulation in C4 photosynthesis: mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin.

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using 14C-iodoacetate and 14C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.

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.

The ferredoxin/thioredoxin system of enzyme regulation in a cyanobacterium

Cell-free preparations of the cyanobacterium (bluegreen alga) Nostoc muscorum were assayed for thioredoxins and enzymes catalyzing the ferredoxin and NADP-linked reduction of thioredoxin. Nostoc was found to have two different thioredoxins: one of approximate molecular weight 16,000 (designated Nostoc thioredoxin f) that selectively activated chloroplast fructose 1,6-bisphosphatase, and another of approximate molecular weight 9,000 (designated Nostoc thioredoxin m) that selcetively activated chloroplast NADP-malate dehydrogenase. The two thioredoxins could be reduced either chemically with dithiothreitol or photochemically with ferredoxin and ferredoxin-thioredoxin reductase which, like the recently found regulatory iron-sulfur protein ferralterin, was present in Nostoc cells. Nostoc ferredoxin-thioredoxin reductase appeared to be similar to its chloroplast counterpart in enzyme specificity, molecular weight, and spectral properties. The Nostoc and spinach chloroplast ferredoxin-thioredoxin reductases as well as their thioredoxins, ferredoxins, and chlorophyll containing membranes were interchangeable in activating chloroplast fructose 1,6-bisphosphatase and NADP-malate dehydrogenase. There was no evidence for an NADP-linked thioredoxin reductase such as that of E. coli. The results are in accord with the conclusion that the cyanobacteria resemble higher plants in having a functional ferredoxin/thioredoxin system rather than an NADP/thioredoxin system typical of other bacteria.

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes.

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5′-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.

Occurrence of cytoplasmic f- and m-type thioredoxins in leaves

There is a growing body of evidence that thioredoxins (proteins that act as regulatory messengers in linking light to enzyme modulation in chloroplasts [ 1,2]) occur in multiple forms in photosynthetic cells [3-61. Two types of thioredoxins are found in chloroplasts (thioredoxins f and m), and a third type is found outside chloroplasts, possibly in the cytoplasm (thioredoxin c) [6]. Chloroplast thioredoxinsfand m are reduced photochemically by chloroplasts via ferredoxin and ferredoxin-thioredoxin reductase or, independently of light, in vitro by the nonphysiological sulfhydryl reagent dithiothreitol. When reduced, thioredoxinf activates specific chloroplast enzymes, including enzymes of the reductive pentose phosphate cycle (fructose-l ,6-bisphosphatase (Fru-Psse), NADPglyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, sedoheptulose-1,7-bisphosphatase [6]) and an enzyme of secondary plant metabolism (phenylalanine ammonia lyase [7]). By contrast, thioredoxin m is specific in its activation of the single chloroplast enzyme, NADP-malate dehydrogenase (NADP-MDH [6]). The function of cytoplasmic thioredoxin c is unknown. In our research, thioredoxin c resembled its chloroplast counterparts in promoting the activation of chloroplast enzymes. However, unlike chloroplast thioredoxins, thioredoxin c appeared to be nonspecific in that when reduced with dithiothreitol it

Activation of plant acid phosphatases by oxidized glutathione and dehydroascorbate

Abstract An acid phosphatase that was partially purified from spinach leaves was activated by oxidized glutathione or dehydroascorbate. Activation was accompanied by a change in the pH optimum so that the enzyme became active in the neutral as well as in the acid region. At neutral pH, the activation induced by oxidized glutathione was reversed by reduced glutathione. Similar results were obtained with a commercial preparation of potato tuber phosphatase. The results suggest that oxidized glutathione and dehydroascorbate serve not only in the previously demonstrated deactivation of thioredoxin-linked enzymes but also in the activation of other enzymes from plants.

Ferredoxin/thioredoxin system functions with effectors in activation of NADP-glyceraldehyde 3-phosphate dehydrogenase of barley seedlings

Abstract NADP-glyceraldehyde 3-phosphate dehydrogenase, a chloroplast enzyme whose activity has been historically linked to greening, was present in an inactive state in barley seedlings germinated in the dark. The inactive enzyme was converted to an active enzyme by effectors such as ATP and by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with the nonphysiological sulfhydryl reagent dithiothreitol. NAD-linked glyceraldehyde 3-phosphate dehydrogenase was not affected by enzyme effectors or thioredoxin under these conditions. The results suggest that NADP-glyceraldehyde 3-phosphate dehydrogenase is present throughout the life cycle of barley plants. The inactive enzyme is converted during greening to an active state by light either via enzyme effectors or via the ferredoxin-thioredoxin system.