A comparative analysis of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system from plants and cyanobacteria

Abstract

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs.

Figure 1.

Immunological characterization of recombinant AnabNTRC and NTR and Trx domains. A, Purified His-tagged (1 μg of protein) OsNTRC (lane 1), AnabNTRC (lane 2), NTR domain (lane 3), or Trx domain (lane 4) of AnabNTRC were subjected to SDS-PAGE under reducing conditions. Proteins were stained with Coomassie Brilliant Blue R-250. Molecular markers were loaded, and their molecular mass, in kD, is indicated on the left. B and C, Replicates of this gel but loaded with 50 ng of the purified proteins were electrotransferred to nitrocellulose membranes and probed with anti-NTRB (B) and anti-OsNTRC (C) antibodies, as indicated.

Figure 2.

Trx and NTR activity of recombinant AnabNTRC and the NTR and Trx domains. A, Insulin reduction catalyzed by the AnabNTRC polypeptide was performed in an incubation mixture containing 2 μm AnabNTRC (squares), 2 μm OsNTRC (diamonds), and 2 μm Trx domain truncated polypeptide (triangles) supplemented with 0.5 mm DTT. B, NADPH-dependent reduction of DTNB was assayed at room temperature in a buffer containing 0.1 μm AnabNTRC (squares), 0.1 μm OsNTRC (diamonds), 0.5 μm NTR domain truncated polypeptide (circles), or 1.0 μm Trx domain truncated polypeptide (triangles) in 100 mm potassium phosphate buffer, pH 7.0, 2 mm EDTA, 5 mm DTNB, and 150 μm NADPH. A negative control in the absence of enzymes was performed (solid line) for both panels. Assays were performed at least three times with similar results, and representative results are shown.

Figure 3.

Reactivity of cyanobacterial and plant NTRC with 2-Cys Prxs from different sources. Assays were performed in reaction mixtures containing 100 mm potassium phosphate buffer, pH 7.0, 2 mm EDTA, 0.25 mm NADPH, 0.5 mm hydrogen peroxide, and purified enzymes at the following concentrations. For A, 4 μm AnabNTRC plus 8 μm 2-Cys Prx from Anabaena (squares), rice (diamonds), and Synechocystis (triangles). Controls were performed with 8 μm Anabaena 2-Cys Prx without AnabNTRC (dotted line) or with 4 μm AnabNTRC without 2-Cys Prx (solid line). For B, 2 μm OsNTRC plus 4 μm 2-Cys Prx from Anabaena (squares), rice (diamonds), and Synechocystis (triangles). Controls were performed with 4 μm rice 2-Cys Prx without OsNTRC (dotted line) or 2 μm OsNTRC without 2-Cys Prx (solid line). Assays were performed at least three times with similar results, and representative results are shown.

Figure 4.

Effect of mutation of the Anabaena NTRC active sites. The activity of the NTRC-2-Cys Prx system was assayed as oxidation of NAD(P)H in a reaction mixture containing 100 mm potassium phosphate buffer, pH 7.0, 2 mm EDTA, 0.5 mm hydrogen peroxide, 8 μm Anabaena 2-Cys Prx, and 2 μm wild-type AnabNTRC supplemented with 0.25 mm NADPH (black squares) or 0.25 mm NADH (white squares). The effect of mutations at the active sites of the NTR and Trx domains of the Anabaena NTRC was assayed replacing the wild-type enzyme by mutants AnabNTRC (C170S) (black triangles) and AnabNTRC (C411S) (white triangles), respectively. Assays were performed at least three times with similar results, and representative results are shown.

Figure 5.

Analysis of the oligomeric state of AnabNTRC and 2-Cys Prx from rice and cyanobacteria. Purified His-tagged AnabNTRC (0.5 mg; A) and purified His-tagged 2-Cys Prx (0.5 mg; B) from rice (thin line), Anabaena (dotted line), and Synechocystis (thick line) were subjected to Superdex 200 gel filtration chromatography in 20 mm potassium phosphate buffer, pH 7.4, 150 mm NaCl. Molecular mass markers, in kD, are indicated.

Figure 6.

Comparison of the structures of 2-Cys Prxs of cyanobacteria, plant, and enteric bacteria. The structural models of 2-Cys Prxs from cyanobacteria (Anabaena and Synechocystis), enteric bacteria (S. typhimurium), and plant (rice) were produced with the PHYRE (http://www.sbg.bio.ic.ac.uk/phyre/html/index.html) modeling server using the following templates: bovine mitochondrial 2-Cys Prx III (c1zyeB) for Anab2CP, human Trx peroxidase B from red blood cells (d1qmva) for Syn2CP, and alkyl hydroperoxide reductase (AhpC) from Helicobacter pylori (c1zofA) for Os2CP. Models were also produced with Swiss-model, obtaining essentially the same results. The positions of Cys residues at the active site are indicated in yellow. The program did not allow the modeling of the C-terminal region of the Anabaena and rice enzymes.

Figure 7.

Analysis of the content of Anabaena NTRC and the redox state of the 2-Cys Prxs in chloroplast stroma from Arabidopsis transgenic lines. Chloroplasts were isolated from leaves of wild-type (WT), ntrc knockout mutant, and independent transgenic lines of Arabidopsis, as indicated. Chloroplasts were lysed by hypoosmotic shock, and the level of endogenous and Anabaena NTRC was analyzed in stromal fractions. A and B, Protein samples (30 μg of protein) were subjected to SDS-PAGE under reducing conditions, blotted onto nitrocellulose membranes, and probed with polyclonal antibodies raised against the rice or Anabaena NTRC, as indicated. C, The redox state of the 2-Cys Prxs was analyzed on protein samples (15 μg of protein), which were fractionated by SDS-PAGE under nonreducing conditions, blotted onto nitrocellulose membranes, and probed with an anti-2-Cys Prx antibody. Band intensities were quantified with the ImageJ software, and the percentage of reduced 2-Cys Prx per line is indicated at the bottom. ox, Oxidized enzyme; red, reduced enzyme.

Figure 8.

Analysis of the complementation of the Arabidopsis ntrc mutant phenotype by expression of the Anabaena NTRC. A, Arabidopsis plants, wild type (WT), ntrc mutant, and transgenic lines expressing either the Arabidopsis or the Anabaena NTRC in the wild-type or mutant background, as indicated, were grown under short-day conditions for 52 d. B to D, Fresh weight (FW) of the rosette leaves (B), content of total leaf chlorophyll (C), and leaf carotenoids (D) were determined. The experiment was repeated three times, and se values are indicated. Values with different letters are significantly different (P ≤ 0.05) as determined by Tukey’s multiple range test.