Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii

Abstract

Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea--strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii--a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes--including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

Keywords: early Earth; evolution; hydrothermal vent; methanogenic archaea; redox regulation.

Fig. 1.

Distribution of thioredoxin homologs in methanogens. A 16S-ribosomal RNA gene-based maximum-likelihood phylogenetic tree constructed as described previously (18) provides a platform for this presentation. Black dots at the branches, confidence values ≥700 (out of 1,000 replicates). Scale bar, number of base substitution per site. The 16S-rRNA gene of Desulfurococcus fermentans (not shown) was used as outgroup. *Abbreviations: IPA and IBA, isopropanol and isobutanol; Me, methanol and mono-, di-, and trimethylamines; Me-H, methanol + H₂; Me-S, dimethylsulfide, and methanethiol. ^†Not detected via BLAST searches.

Fig. 2.

Activation of F₄₂₀-dependent sulfite reductase or Fsr and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase or Mtd by Trx1. Fsr and Mtd were preincubated with Trx1, DTT, or both at 65 °C for 5 min followed by an additional incubation at 25 °C for 20 min and then assayed for activity. Enzyme without a treatment was used as the control. Solid bar, an average of values from replicates (three independent experiments for Fsr and two for Mtd). Error bar, SD. Number on a solid bar, fold of activation. Label below a bar, reagents used for treatment.

Fig. 3.

Select reactions and pathways of M. jannaschii targeted by Trx1 (Mj_0307). The methanogenesis pathway was redrawn from ref. . Color codes: red and green, enzymes identified as Trx1 targets in two or more and one experiment(s), respectively; blue, not targeted by Trx1. The dashed arrows show extended biosynthetic routes. 1,3-BPG, 1,3-bisphosphoglycerate; [CO], enzyme-bound carbon monoxide (CO); CoB, coenzyme B; CoM, coenzyme M; DHAP, dihydroxyacetone phosphate; Ech, energy-converting hydrogenase; F₄₂₀, coenzyme F₄₂₀; FBP aldolase, fructose bisphosphate aldolase; FBPase, fructose bisphosphatase; *Fd, specific ferredoxin; Frd, fumarate reductase; Ftr, formylmethanofuran-H₄MPT formyltransferase; Fwd and Fmd, tungsten- and molybdenum-dependent formylmethanofuran dehydrogenase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; GD3P, glyceraldehyde-3-phosphate; H₄MPT, tetrahydromethanopterin; Hdr-H₂ase, electron-bifurcating hydrogenase-heterodisulfidereductase complex; HS-CoA, CoA; α-Kgfor and Pfor, α-ketoglutarate- and pyruvate-ferredoxin oxidoreductase; Mch, methenyl-H₄MPT cyclohydrolase; Mcr, methyl-coenzyme M reductase; Mdh, malate dehydrogenase; Mer, methylene-H₄MPT reductase; MF, methanofuran; Mtd and Hmd, F₄₂₀- and H₂-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-H₄MPT-coenzyme M methyltransferase; Δμ Na⁺, electrochemical sodium ion potential; PEP, phosphoenolpyruvate; 3-PG and 2-PG, 3- and 2-phosphoglycerate; Pgi, phosphoglycerate isomerase; Pgk, phosphoglycerate kinase; 2-Pgm, 2-phosphoglycerate mutase; Pps, phospoenolpyruvate synthase; Pyc, pyruvate carboxylase; Sdh, succinate dehydrogenase; Tpi, triose phosphate isomerase.