title: 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.
authors.affiliation: Department of Biological Sciences, University of Arkansas-Fayetteville, AR, USA.
authors.forename: Addison C
authors.initials: AC
authors.lastname: McCarver
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Lessner
mesh_terms: D003001:Cloning, Molecular; D004579:Electron Transport; D017020:Methanosarcina / Q000254:growth & development / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D013879:Thioredoxins / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase
keywords: anaerobe; archaea; methanogen; thioredoxin; thioredoxin reductase
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