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Thioredoxin Targets Fundamental Processes in a Methane-Producing Archaeon, Methanocaldococcus Jannaschii

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Thioredoxin Targets Fundamental Processes in a Methane-Producing Archaeon, Methanocaldococcus Jannaschii Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii Dwi Susantia,b,c, Joshua H. Wongd, William H. Vensele, Usha Loganathana,c,f, Rebecca DeSantisg,1, Ruth A. Schmitzg, Monica Balserah, Bob B. Buchanand,2, and Biswarup Mukhopadhyaya,c,f,2 Departments of aBiochemistry and fBiological Sciences, bGenetics, Bioinformatics and Computational Biology Graduate Program, and cVirginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061; dDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720; eWestern Regional Research Center, United States Department of Agriculture, Agricultural Research Service, Albany, CA 94710; gInstitut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany; and hDepartamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), 37008 Salamanca, Spain Contributed by Bob B. Buchanan, January 7, 2014 (sent for review September 10, 2013) Thioredoxin (Trx), a small redox protein, controls multiple pro- strict anaerobes that produce methane, a prominent greenhouse cesses in eukaryotes and bacteria by changing the thiol redox gas and important fuel. We have focused on Methanocaldococcus status of selected proteins. The function of Trx in archaea is, jannaschii—a deeply rooted, hyperthermophilic methanogen living however, unexplored. To help fill this gap, we have investigated in deep-sea hydrothermal vents (10) where conditions mimic those this aspect in methanarchaea—strict anaerobes that produce meth- of early Earth. M. jannaschii produces methane exclusively from ane, a fuel and greenhouse gas. Bioinformatic analyses suggested H2 and CO2 via a process believed to represent an ancient form of that Trx is nearly universal in methanogens. Ancient methanogens respiration (11). M. jannaschii thus presents an opportunity to that produce methane almost exclusively from H2 plus CO2 carried explore the role of Trx in an archaeon and, at the same time, gain approximately two Trx homologs, whereas nutritionally versatile insight into the evolutionary history of redox regulation. Our members possessed four to eight. Due to its simplicity, we studied results suggest that Trx alleviates oxidative stress in methanogens the Trx system of Methanocaldococcus jannaschii—a deeply rooted via a thiol-based mechanism that could also regulate fundamental hyperthermophilic methanogen growing only on H2 plus CO2.The processes by redox transitions in the absence of O2.Therole organism carried two Trx homologs, canonical Trx1 that reduced formulated for this anaerobic archaeon confirms and extends insulin and accepted electrons from Escherichia coli thioredoxin re- that established for aerobic forms of life. ductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets Results representing a range of processes—including methanogenesis, biosyn- Thioredoxin Homologs of Methanarchaea. Iterative BLAST searches thesis, transcription, translation, and oxidative response. In enzyme (12) using Escherichia coli and M. jannaschii Trxs as queries and assays, Trx1 activated two selected targets following partial deactiva- screening output for hits with the C-X-X-C motif and appropriate tion by O2, validating proteomics observations: methylenetetrahydro- sizes of 70- to 110-aa residues (13) showed that Trx homologs exist methanopterin dehydrogenase, a methanogenesis enzyme, and sul- in almost all methanogen genomes represented in the National fite reductase, a detoxification enzyme. The results suggest that Trx Center for Biotechnology Information (NCBI) database (Fig. 1 assists methanogens in combating oxidative stress and synchroniz- and Table S1). Methanopyrus kandleri AV19, a hydrothermal ing metabolic activities with availability of reductant, making it a crit- vent-associated hyperthermophilic methanogen (optimum growth ical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it Significance 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. This study extends thioredoxin (Trx)-based oxidative redox regulation to the archaea, the third domain of life. Our study methanogenic archaea | redox regulation | hydrothermal vent | suggests that Trx is nearly ubiquitous in anaerobic metha- early Earth | evolution nogens, enabling them to recover from oxidative stress and synchronize cellular processes, including methane biogenesis, ∼ with the availability of reductants. As methane is a valuable hioredoxins (Trxs) are small ( 12-kDa) redox proteins typi- fuel, an end product of anaerobic biodegradation and a potent Tcally bearing a characteristic Cys-Gly-Pro-Cys motif that re- greenhouse gas, Trx may now be considered a critical partici- duce specific disulfide bonds of selected proteins (1). Reduction pant in the global carbon cycle, climate change, and bioenergy — alters the biochemical properties of the proteins targeted e.g., by production. Because methanogenesis developed before the increasing their activity or solubility (1). Trxs are found in the three oxygenation of the earth, our work raises the possibility that domains of life: bacteria, eukarya, and archaea (2). In eukarya and Trx functioned in a complex redox regulatory network in an- bacteria, the regulatory role of Trx has been shown to span the aerobic prokaryotes at least 2.5 billion years ago. major aspects of metabolism, including photosynthesis, biosynthesis, replication, transcription, translation, and stress response (1). Trx Author contributions: D.S., J.H.W., W.H.V., R.A.S., M.B., B.B.B., and B.M. designed research; D.S., also acts as an electron donor for enzymes, notably ribonucleotide J.H.W., W.H.V., U.L., and R.D. performed research; D.S., J.H.W., W.H.V., R.A.S., M.B., B.B.B., and B.M. analyzed data; and D.S., J.H.W., W.H.V., B.B.B., and B.M. wrote the paper. reductase, phosphoadenosinephosphosulfate reductase, methionine The authors declare no conflict of interest. sulfoxide reductase, and peroxiredoxins (1). However, in contrast 1Present address: Department of Intensive Care and Intermediate Care, University Hospital, to the wealth of information for bacteria and eukaryotes, our un- Rheinisch-Westfaelische Technische Hochschule Aachen University, 52074 Aachen, Germany. derstanding of archaeal Trx is limited to its biochemical and struc- 2To whom correspondence may be addressed. E-mail: [email protected] or view@ tural properties (3–9). Its physiological role remains a mystery. berkeley.edu. To help fill this gap, we have investigated the role of Trx in a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. group of archaea known as methanogens or methanarchaea— 1073/pnas.1324240111/-/DCSupplemental. 2608–2613 | PNAS | February 18, 2014 | vol. 111 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1324240111 Downloaded by guest on September 25, 2021 temperature, 98 °C), was apparently the only exception in lacking a were oxidized by aerobic dialysis, and the remaining free sulfhydryl recognizable homolog of Trx (14). groups of the air-exposed proteins were blocked by alkylation. The Methanococci and Methanobacteria carried an average of two Trx extract was then treated with Trx1 using either DTT or NADPH homologs, with their numbers ranging from one to four, whereas (plus E. coli NTR) as reductant, anticipating that Trx1 would re- Methanomicrobia possessed two to eight Trx homologs, with an duce the regulatory disulfide (S–S) groups formed in aerobic di- average of four. Methanocorpuscullum labreanum, amemberofthe alysis. The newly available free –SH groups were derivatized with the latter class, was an exception in possessing two Trx homologs. fluorescent probe monobromobimane (mBBr), and the labeled proteins were resolved in 2D gels (Fig. S2 A and B). The fluorescent Trxs of M. jannaschii. M. jannaschii (Mj) carries two Trx homologs, spots, which were either absent or less intense in control gels, were Mj_0307 and Mj_0581 (9, 15), here called Trx1 and Trx2, re- analyzed by mass spectrometry (17). The experiment with DTT was spectively. The sequence identity and similarity between Trx1 performed in triplicate and that with Ec-NTR+NAPDH was per- and Trx2 are 23% and 49%, respectively. Both proteins have formed once. From these experiments, we identified a total of 152 homologs in Methanothermobacter thermautotrophicus ΔH (7, 8), potential Trx1 targets (Table 1 and Table S2). Of these, 19 proteins where Trx1 is closely related to MTH807 (identity, 51%; simi- were identified in all four experiments, and 18, 38, and 77 were larity, 67%) and Trx2 corresponds to MTH895 (identity, 37%; detected in three, two, and one of the experiments, respectively. similarity, 54%). Purified recombinant Trx1 and Trx2 were re- duced by dithiothreitol (DTT) (Fig. S1A). However, the proteins Effect of Reduction by Trx1 on the Activity of Selected M. jannaschii were distinct in two well-characterized activities in which Trx1 Enzymes. F420-dependent sulfite reductase. An air-exposed 7,8-dide- exhibited a closer resemblance to E. coli Trx, a standard in the field. methyl-8-hydroxy-5-deazaflavin-5′-phosphoryllactyl glutamate [co- First, in the insulin reduction assay, Trx1 showed 80-fold higher enzyme F420 (F420)]-dependent sulfite
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