Molecular characterization of the thioredoxin system from Methanosarcina acetivorans

Abstract

The thioredoxin system, composed of thioredoxin reductase (TrxR) and thioredoxin (Trx), is widely distributed in nature, where it serves key roles in electron transfer and in the defense against oxidative stress. Although recent evidence reveals Trx homologues are almost universally present among the methane-producing archaea (methanogens), a complete thioredoxin system has not been characterized from any methanogen. We examined the phylogeny of Trx homologues among methanogens and characterized the thioredoxin system from Methanosarcina acetivorans. Phylogenetic analysis of Trx homologues from methanogens revealed eight clades, with one clade containing Trxs broadly distributed among methanogens. The Methanococci and Methanobacteria each contain one additional Trx from another clade, respectively, whereas the Methanomicrobia contain an additional five distinct Trxs. Methanosarcina acetivorans, a member of the Methanomicrobia, contains a single TrxR (MaTrxR) and seven Trx homologues (MaTrx1-7), with representatives from five of the methanogen Trx clades. Purified recombinant MaTrxR had 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase and oxidase activities. The apparent Km value for NADPH was 115-fold lower than that for NADH, consistent with NADPH as the physiological electron donor to MaTrxR. Purified recombinant MaTrx2, MaTrx6 and MaTrx7 exhibited dithiothreitol- and lipoamide-dependent insulin disulfide reductase activities. However, only MaTrx7, which is encoded adjacent to MaTrxR, could serve as a redox partner to MaTrxR. These results reveal that M. acetivorans harbors at least three functional and distinct Trxs, and a complete thioredoxin system composed of NADPH, MaTrxR and at least MaTrx7. This is the first characterization of a complete thioredoxin system from a methanogen, which provides a foundation to understand the system in methanogens.

Keywords: anaerobe; archaea; methanogen; thioredoxin; thioredoxin reductase.

Fig. 1. Phylogenetic analyses of methanogen Trx homologues

A simplified phylogenetic tree based on the complete tree (Fig. S1). Clades (A–H) are labelled and differently colored. Numbers above nodes represent maximum-likelihood bootstrap values; only values >50% are shown.

Fig. 2. SDS-PAGE analysis of recombinant proteins purified from E. coli

The purified recombinant proteins (3 µg each) were separated on 15% SDS-PAGE. MW, Marker lane.

Fig. 3. Spectroscopic analysis of MaTrxR

(A) UV-visible spectrum of 10 µM as-purified MaTrxR (gray line) and FAD-reconstituted MaTrxR (black line). Inset: magnified spectrum of as-purified MaTrxR. (B) Spectrum of 6.3 µM MaTrxR before (black line) and after (gray line) the addition of 70 µM NADPH under anaerobic conditions. (C) Spectrum of 6.3 µM MaTrxR before (black line) and after (gray line) the addition of 110 µM NADH under anaerobic conditions. All spectra were of MaTrxR in 50 mM Tris-HCl pH 7.5, 150 mM NaCl.

Fig. 4. Comparison of the reduction of insulin catalyzed by MaTrxs

(A) DTT-dependent activity: 9 µM MaTrx2 (triangles), 3 µM MaTrx6ΔSp (squares), and 6 µM MaTrx7 (diamonds) were added to 0.33 mM DTT and 0.13 mM insulin in 100 mM KPO₄ pH 6.8 under anaerobic conditions. The complete reaction without the addition of thioredoxin was included as a negative control (circles). Absorbance at 650 nm at 2 min intervals is shown. The data are the mean ± SD of triplicate reactions and the specific activity (ΔA650/min² × 10⁻³/mg) of each thioredoxin is shown in the inset. (B) Lipoamide-dependent activity: 12 µM MaTrx2 (triangles), 3 µM MaTrx6ΔSp (squares), and 6 µM MaTrx7 (diamonds) were added to 0.33 mM NADH, 4 units lipoamide dehydrogenase, 0.05 mM lipoamide and 0.13 mM insulin in 100 mM KPO₄ pH 6.8 under anaerobic conditions. The complete reaction without the addition of thioredoxin was included as a negative control (circles). Absorbance at 650 nm at 4 min intervals is shown. The data are the mean ± SD of triplicate reactions and the specific activity (ΔA650/min² × 10⁻³/mg) of thioredoxin is shown in the inset.

Fig. 5. Comparison of the reduction of insulin catalyzed by the M. acetivorans thioredoxin system components

(A) NADPH-dependent activity: 10 µM MaTrx2, 10 µM MaTrx6ΔSp, 5 µM MaTrx7, or 10 µM EcTrx1 were added to 0.35 mM NADPH, 1 µM MaTrxR, and 0.13 mM insulin in 100 mM KPO₄ pH 6.8 under anaerobic conditions. The complete reaction without the addition of thioredoxin was included as a negative control (not shown). Absorbance at 650 nm at 4 min intervals is shown. The data are the mean of triplicate reactions. (B) NADH-dependent activity: 10 µM MaTrx2, 10 µM MaTrx6ΔSp, 5 µM MaTrx7, or 10 µM EcTrx1 were added to 1 mM NADH, 1 µM MaTrxR, and 0.13 mM insulin in 100 mM KPO4 pH 6.8 under anaerobic conditions. The complete reaction without the addition of thioredoxin was included as a negative control (not shown). Absorbance at 650 nm at 4 min intervals is shown. The data are the mean of triplicate reactions.