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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.

SUBMITTER: McCarver AC

PROVIDER: S-EPMC4199913 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Molecular characterization of the thioredoxin system from Methanosarcina acetivorans.

McCarver Addison C AC Lessner Daniel J DJ

The FEBS journal 20140906 20

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 ...[more]

PMID: 25112424

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Project description:Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

Project description:BackgroundAcetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H₂ as an intermediate, although the great majority of acetotrophs are unable to metabolize H₂. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H₂-utilizing Methanosarcina acetivorans from H₂-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.ResultsA component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.ConclusionsThe results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H₂-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

Dataset Information

Molecular characterization of the thioredoxin system from Methanosarcina acetivorans.

Publications

Molecular characterization of the thioredoxin system from Methanosarcina acetivorans.

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