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 ECOLOGY 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.

www.pnas.org/cgi/doi/10.1073/pnas.1324240111 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 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 carriedanaverageoftwoTrx 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 reductase (Fsr) preparation activity than Trx2, and Trx2 exhibited a longer lag, 35 vs. 10 min for showed two-thirds the activity observed with the corresponding Trx1 (Fig. S1B). Second, Trx1 but not Trx2 was reduced by E. coli anaerobic preparation, the respective values being 0.132 and 0.200 nicotinamide adenine dinucleotide phosphate (NADP)-thioredoxin U/mg. Assay of oxidized Fsr with Trx1 (20 μM) at 65 °C in the reductase (Ec-NTR) with NADPH. It is noteworthy that the ho- presence of 1 mM DTT increased the activity of the enzyme by 2.9- molog of Trx2, M. thermautotrophicus ΔH (MTH895), unlike the M. fold (Fig. 2); with twice as much Trx1, activation was 4.6-fold. DTT jannaschii protein, accepts electrons from E. coli NTR (8). alone (1 mM) inhibited the enzyme, and Trx1 alone activated Fsr 1.5-fold, likely due to a protein concentration effect. Identification of Trx1 Targets. A fluorescent gel/proteomics ap- F420-dependent methylenetetrahydromethanopterin dehydrogenase. Non- proach that proved successful in several plant investigations (1, 16) reducing SDS/PAGE developed with a monobromobimane (mBBr)- wasusedtoidentifytheM. jannaschii proteins reduced by Trx1 treated preparation revealed that purified recombinant F420- (Trx1 targets). Briefly, in this procedure, M. jannaschii cell extracts dependent methylenetetrahydromethanopterin dehydrogenase

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, con- fidence 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, meth-

anol + H2; Me-S, dimethylsulfide, and methanethiol. †Not detected via BLAST searches.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1324240111 Susanti et al. Downloaded by guest on October 2, 2021 Table 1. Potential M. jannaschii Trx1 targets Metabolic function or structural unit Potential targets

ATP synthesis V-type ATP synthase subunit A*, V-type ATP synthase subunit B* Biosynthesis Capsular polysaccharide biosynthesis protein, GMP synthase II, inosine-5′-monophosphate dehydrogenase I, orotate phosphoribosyltransferase-like protein, phosphoribosylaminoimidazole synthetase, phosphoribosylaminoimidazole-succinocarboxamide synthase, phosphoribosylformylglycinamidine synthase II, pyridoxal biosynthesis lyase PdxS, ribose-phosphate pyrophosphokinase, spermidine synthase*, uridylate kinase* Coenzyme M biosynthesis Phosphosulfolactate synthase Defense against Csm3 family CRISPR-associated RAMP protein foreign DNA Hypothetical protein Hypothetical protein MJ_0164, hypothetical protein MJ_0308, hypothetical protein MJ_1099 Metabolism 2-Oxoglutarate ferredoxin oxidoreductase subunit γ, acetyl-CoA decarbonylase/synthase complex subunit γ, fructose-bisphosphate aldolase*, fructose-1,6-bisphosphatase*, phosphoenolpyruvate synthase, phosphopyruvate hydratase (enolase)*, putative transaldolase*, pyruvate carboxylase subunit B, pyruvate ferredoxin oxidoreductase subunit α PorA*, UDP-glucose dehydrogenase*

Methanogenesis F420-dependent methylenetetrahydromethanopterin dehydrogenase, (energy generation) formylmethanofuran–tetrahydromethanopterin

formyltransferase, H2-dependent methylenetetrahydromethanopterin

dehydrogenase, H2-dependent methylenetetrahydromethanopterin dehydrogenase-like + protein I, H -transporting ATP synthase subunit E AtpE, methyl coenzyme M reductase I subunit McrA, methyl coenzyme M reductase I subunit McrB, methylenetetrahydromethanopterin

reductase, methylviologen-reducing hydrogenase subunit α, N5,N10-methenyltetrahydromethanopterin

cyclohydrolase ECOLOGY Miscellaneous AMMECR 1 domain protein, methanogenesis marker protein 17, methyltransferase, iron-sulfur flavoprotein Nitrogen and amino (R)-2-Hydroxyglutaryl-CoA dehydratase activator, 2-hydroxyglutaryl-CoA dehydratase, acid metabolism 3-dehydroquinate synthase, acetolactate synthase catalytic subunit*, anthranilate synthase component II TrpD, argininosuccinate synthase*, aspartate aminotransferase*, aspartate-semialdehyde dehydrogenase*, branched-chain amino acid aminotransferase, D-3-phosphoglycerate dehydrogenase*, dihydrodipicolinate reductase, dihydrodipicolinate synthase, dihydroxy-acid dehydratase*, ketol-acid reductoisomerase*, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide, -isomerase HisA1, S-adenosylmethionine synthetase* Oxidative stress response Flavoprotein FpaA, NADH oxidase, peroxiredoxin* Replication, transcription, 30S ribosomal protein S7, 50S ribosomal protein L6, acidic ribosomal protein P0, arginyl-tRNA and translation synthetase, cell division protein CDC48*, cell division protein FtsZ I*, elongation factor 1-α*, elongation factor EF-2, thermosome Structural proteins Flagella-like protein E, S-layer protein † Sulfite detoxification F420-dependent sulfite reductase Transport proteins High-affinity branched-chain amino acid transport protein BraC

Targets that were identified in at least two independent experiments are reported here. A more extensive list appears in Table S2. *Previously identified as Trx target in eukaryotic and bacterial systems. † A non–F420-dependent sulfite reductase has been identified as a Trx target in certain bacteria and eukaryotes.

(Mtd) was recovered mostly in reduced form. To generate po- carried a limited number of Trx homologs (two to four; two on tential redox active cystine disulfides, the enzyme was treated average). These organisms have relatively smaller genomes (1.24– with several oxidants, H2O2, CuCl2, and Aldrithiol-2. Based on 2.94 Mbp; NCBI data), include almost all hyperthermophilic or mBBr gel analysis, Aldrithiol-2 proved most effective in oxidizing thermophilic methanogens (19), and are mostly restricted to H2- Mtd. This oxidation deactivated the enzyme by 53%. A treat- dependent methanogenesis (19). By contrast, the late-evolving ment of the deactivated enzyme with fivefold molar excess Trx1 Methanomicrobia with larger genomes (1.8–5.75 Mbp; NCBI and 0.05 mM DTT yielded a 4.4-fold increase in activity vs. a 1.4- data) and more complex metabolism carried up to eight Trxs (on fold enhancement seen with DTT alone (Fig. 2). average, four). The methylotrophic Methanomicrobia use meth- anol and methylamines, and some of these perform methano- Discussion genesis from acetate as well as H2 and CO2 (19, 20); most are Distribution of Trx Homologs in Methanogens. The evidence pre- mesophiles and relatively O2 tolerant. sented above suggests that Trx homologs are nearly universal in It is possible that the Trx system came into play in the deeply methanogenic archaea. M. kandleri, the most deeply rooted and rooted methanogenic archaea as these organisms faced a more the most thermophilic methanogen known (growth occurs at oxidizing environment brought about by H2 limitation or O2 84–110 °C), was the only exception. This organism, which is exposure. It remains to be seen whether the larger number of Trxs solely dependent on H2 and CO2 for methanogenesis (14), in late-evolving methanogens is a result of horizontal gene transfer lacked a recognizable homolog of Trx. In the other metha- or gene duplication coupled with subsequent diversification. We nogens, the distribution of Trx homologs followed a pattern (Fig. note that these organisms’ ability to use a range of methanogenic 1 and Table S1). Phylogenetically deeply rooted representatives substrates is thought to be due to a large number of genes ac- belonging to the classes of Methanococci and Methanobacteria quired from the Clostridia and other anaerobes (21).

Susanti et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 archaeon may use a similar mode of action of Trx to regulate the activity of selected enzymes, thereby synchronizing metabolism with the availability of reductant such as H2 under normal an- aerobic conditions. In the deep-sea hydrothermal vents M. jan- naschii inhabits, changes in partial pressure of H2 can be extreme (4 Pa to 200 kPa) (19, 25, 27, 28) and exposure to O2 may occur following the entry of aerobic seawater (27, 28). The expression of several methanogenesis-related genes in M. jannaschii and other H2-oxidizing methanogens is transcrip- tionally regulated by H2 availability, and our observations suggest the presence of parallel posttranslational control effected by Trx Fig. 2. Activation of F420-dependent sulfite reductase or Fsr and F420- (Table 1 and Table S2; Fig. 2). This possibility is also consistent dependent methylenetetrahydromethanopterin dehydrogenase or Mtd by with the proposal that Mtd is the primary enzyme for the re- Trx1. Fsr and Mtd were preincubated with Trx1, DTT, or both at 65 °C for duction of methylenetetrahydromethanopterin under H2 limita- 5 min followed by an additional incubation at 25 °C for 20 min and then tion (29, 30) and with our finding of a direct effect of Trx on the assayed for activity. Enzyme without a treatment was used as the control. activity of the enzyme. Significantly, the alternate H -dependent Solid bar, an average of values from replicates (three independent experi- 2 enzyme [H2-dependent methylenetetrahydromethanopterin de- ments for Fsr and two for Mtd). Error bar, SD. Number on a solid bar, fold of hydrogenase (Hmd)], also a potential Trx1 target, is active under activation. Label below a bar, reagents used for treatment. high H2 partial pressure (31). In view of these results, it is timely to determine whether Trx regulates the activity of Hmd and M. jannaschii Trxs. Trx1 and Trx2 were distinct in terms of amino other methanogenesis enzymes identified as targets (Table 1 and acid sequence, reactivity with insulin, and activity with E. coli Table S2) as well as counterparts in terrestrial systems where O2 NTR. These features are possibly related to the nature of the exposure and changes in reductant supply are common (19, 20). putative redox active site motif C-X-X-C as the two internal The ability to down-regulate methanogenesis, the sole avenue for residues (X’s) influence the redox properties of the protein (22). energy generation, via the oxidation of sulfhydryl groups would In Trx1 and Trx2, this motif is, respectively, C-P-H-C and C-P-K-C, enable methanogens to attain a dormant-type state. With the return which differ from each other and from the classical C-G-P-C (21). It of favorable environmental conditions, Trx could reactivate target is thus not surprising that Trx1 and Trx2 showed different spe- methanogenesis enzymes via disulfide reduction and thereby restore cificities. Trx1 was typical—i.e., similar to its E. coli counterpart in growth and other vital cell processes. This situation resembles the primary structure, robust insulin reduction activity, and reduction role of Trx in initiating processes associated with seed germination by E. coli NTR. This protein was, therefore, chosen for identifying (32). Modification of F420 via adenylation and guanylation provides candidate Trx target proteins in M. jannaschii. an alternate avenue for enabling a methanogen to shut down energy production and achieve a dormant state (33). Targets of Trx1. Proteomics and enzyme activity measurements Sulfite detoxification. Identified as a target in proteomics studies suggested that Trx1 influences multiple processes in M. jannaschii, (Table S2), Fsr was deactivated upon exposure to O2, and the including methanogenesis—the hallmark of the methanogens. Our altered enzyme was partially reactivated by reduced Trx1 (Fig. 2). proteomics analysis revealed a total of 152 M. jannaschii poly- These observations make physiological sense. Sulfite inhibits peptides as potential Trx1 targets, representing ∼10% of the total methyl-coenzyme M reductase and, thereby, impedes methano- ORFs in the organism’s genome (Table S2). Of these, 75 targets genesis (25). In the habitat of M. jannaschii, sulfite is formed when were detected in at least two of four independent experiments O2-containing cold seawater mixes with the hot sulfide-rich vent (Table 1) and more than one-half were observed only once. As fluid (25). Fsr detoxifies the newly formed sulfite by reducing it to shown in Table S2, most of the targets contain at least two Cys sulfide, an essential nutrient for methanogens (25). Because sul- residues, indicative of Trx-reducible intramolecular or intermol- fite can oxidize protein sulfhydryl (SH) groups to the disulfide ecular Cys disulfide bonds (Table S2). Curiously, a few of the (S–S) level (34), it is not surprising that oxidatively deactivated Fsr targets have only one Cys, raising the possibility that in these can be reductively activated by Trx1. It is possible that activation by instances Trx reduces intermolecular disulfides as described for Trx is a general feature of sulfite reductases as the enzyme of wheat yeast 1-Cys peroxiredoxin (23). The putative peroxiredoxin of M. starchy endosperm appears also to be a Trx target (35). jannaschii (MJ_0736), however, contains five Cys. Biosynthesis. The de novo synthesis of acetate and pyruvate from The Trx1 target proteins participate in multiple processes in CO2 are key initial anabolic steps for an autotroph such as addition to methanogenesis: biosynthesis, information process- M. jannaschii (10). It is significant that the two enzymes of this ing, cell division, sulfite detoxification, oxidative response, and process, acetyl-CoA decarbonylase/synthase (ACDS) and pyru- resistance to phages and invasion by foreign DNA. Structural vate:ferredoxin oxidoreductase (PFOR), were both identified as proteins were also identified as Trx1 targets. The results reveal Trx1 targets (Table 1 and Fig. 3). Trx is known to revive oxida- the obvious vulnerability of an ancient methanogen cell to oxidative tively damaged PFOR in Desulfovibrio africanus,ananaerobic stress and the suitability of Trx for repairing the resulting damage. sulfate reducing bacterium (36, 37). However, the situation may be To confirm and extend the proteomics results, we tested the ef- different in M. jannaschii as PFOR and ACDS of methanogens fect of reduced Trx1 on the in vitro activity of two candidate target have been reported to be irreversibly inactivated by O2 exposure in enzymes: Mtd, a core enzyme of the methanogenesis pathway (24), vitro (38, 39). In methanogens, the role of Trx could lie in recovery and Fsr (25), an enzyme that enables certain methanogens to tol- from less severe O2 exposure or in redox regulation of activity. erate and use sulfite as a source of sulfur. Oxidized forms of both Methanogens could invoke the latter in response to a drop in H2 enzymes were activated by Trx1, giving further credence to the partial pressure—a fact of life in most, if not all, of their natural fluorescent/gel approach of target identification. Mtd and Fsr were habitats (19, 20, 25, 27, 28). In these cases, the prevailing midpoint + selected based on two criteria: being specific to methanarchaea and potential values of H /H2 redox couple would make the formation the availability of assay tools in our laboratories. of protein disulfides thermodynamically feasible. Because the level of coenzyme F420 in a methanogen cell is in equilibrium with M. jannaschii Systems Targeted by Trx1. Methanogenesis. It is signif- environmental H2 partial pressure (40, 41), this mechanism of icant that many of the enzymes identified as Trx targets function in sulfhydryl oxidation could be performed by an F420-dependent the reduction of CO2 to CH4 (Fig. 3, Table 1, and Table S2). enzyme acting directly or via an intermediary. Based on the nature of our experiments, this observation suggests The synthesis of sugars via gluconeogenesis is an energy- that, reminiscent of plants, Trx activates enzymes of M. jannaschii intensive pathway that requires a reductant. In eukarya, many of that have been deactivated following O2 exposure (1, 26). The the associated enzymes are linked to Trx (26). Significantly,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1324240111 Susanti et al. Downloaded by guest on October 2, 2021 Fig. 3. Select reactions and pathways of M. jannaschii targeted by Trx1 (Mj_0307). The methanogenesis path- way was redrawn from ref. 23. 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, co- enzyme B; CoM, coenzyme M; DHAP, dihydroxyacetone

phosphate; Ech, energy-converting hydrogenase; F420, coenzyme F420; FBP aldolase, fructose bisphosphate aldolase; FBPase, fructose bisphosphatase; *Fd, spe- cific ferredoxin; Frd, fumarate reductase; Ftr, for-

mylmethanofuran-H4MPT formyltransferase; Fwd and Fmd, tungsten- and molybdenum-dependent formylmethanofuran dehydrogenase; Gapdh, glyceral- dehyde-3-phosphate dehydrogenase; GD3P, glyceralde-

hyde-3-phosphate; H4MPT, tetrahydromethanopterin; Hdr-H2ase, electron-bifurcating hydrogenase-hetero- disulfidereductase complex; HS-CoA, CoA; α-Kgfor and Pfor, α-ketoglutarate- and pyruvate-ferredoxin oxido-

reductase; Mch, methenyl-H4MPT cyclohydrolase; Mcr, methyl-coenzyme M reductase; Mdh, malate de-

hydrogenase; Mer, methylene-H4MPT reductase; MF, methanofuran; Mtd and Hmd, F420-andH2-dependent methylene-H4MPT dehydrogenase; Mtr, methyl-H4MPT- + coenzyme M methyltransferase; Δμ Na , electro- chemical sodium ion potential; PEP, phosphoenol- pyruvate; 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. ECOLOGY

several of their M. jannaschii counterparts were also identified as and Table S2). CRISPR elements and associated proteins pro- potential Trx targets (Fig. 3; Table 1 and Table S2), suggesting vide defense against invasion by external DNA materials such that gluconeogenesis could also be redox regulated in this or- as plasmids and phage in archaea as well as bacteria (47), ganism. Following this same theme, several enzymes of amino raising the possibility that this process is regulated by Trx in acid biosynthesis reported to be Trx targets in plants (26)— M. jannaschii. notably, glutamine synthetase, threonine synthase, and aspartate semialdehyde dehydrogenase were reduced by Trx1 in M. jannaschii Concluding Remarks extracts. Phosphosulfolactate synthase, an enzyme needed for the The present work extends Trx-based redox transition to the third biosynthesis of coenzyme M—a requirement for methane formation domain of life. The methanarchaeon M. jannaschii was found to with all substrates (42), also appeared to be linked to Trx1 (42). A use this protein to protect a range of cellular processes against role in coenzyme M synthesis falls within the broader function of oxidative damage. Interestingly, many of the Trx targets identi- Trx in the repair and regulation of the methanogenesis process. fied have counterparts in plants where an oxidative type of reg- Transcription, translation, and cell division. Transcription and trans- ulation is known to occur (16, 48). lation have previously been linked to Trx-based regulation in The present findings have far-reaching implications to our Bacteria and Eukarya (1). Modification of the RNA polymerase understanding of the evolution of redox regulation as well as ω subunit and elongation factors in E. coli (43) and several to areas of current societal interest. Because M. jannaschii chloroplast ribosomal proteins fall in this category (26). Similar performs hydrogenotrophic methanogenesis, a process that de- controls likely exist in M. jannaschii where a ribosomal protein S7 veloped before the appearance of O2 (11, 49), Trx may have and several tRNA synthetases were identified as Trx1 targets originally functioned in an anaerobic regulatory capacity in this (Table 1 and Table S2). ancient organism. Its participation in protecting cells against O2 Like its counterparts in chloroplasts and E. coli (16, 43), FtsZ, would have developed later. Accordingly, the redox network a cytoskeletal protein similar to tubulin in eukaryotes (44), was created by Trx, and the attendant cellular complexity, would have reduced by Trx1 in M. jannaschii (Table 1 and Table S2). Thus, as developed in prokaryotes at least 2.5 billion years ago. Future with E. coli, Trx acting through FtsZ could contribute to the research will be directed toward this question. On the pragmatic regulation of cell division in this organism. side, due to the role of methanogens in producing methane and Structural proteins. One of the two S-layer proteins, which are the attendant changes in the biosphere, Trx emerges as a key major cell envelope components in methanogens (45), was re- participant in the global carbon cycle, climate change, and duced by Trx1. Interestingly, the level of S-layer protein decreases bioenergy production. Methanococcus maripaludis under H2 limitation in , a close relative Materials and Methods of M. jannaschii (46). Considering that Trx is a posttranslational M. jannaschii modifier, it is possible that both the generation and assembly of Purified Preparations of Trxs, Mtd, and Fsr, and Methanogen the S-layer is redox controlled in methanogens. Cofactors, and Insulin Reduction Assay. Previously described methods were used for generating homogeneous preparations of recombinant His-tagged Defense against reactive oxygen species and foreign DNA. Similar to Trx and Mtd (50) and F420 (51), partial purification of Fsr from M. jannaschii chloroplasts (16), a peroxiredoxin was identified as a Trx target cell extracts (25), and the insulin assay for Trx (52). in M. jannaschii (Table 1 and Table S2). Peroxiredoxins are critical antioxidant enzymes catalyzing the reduction of hydro- Trx-Mediated Reduction of M. jannaschii Cell Extract Proteins, 2D Gel Electrophoresis, peroxides and alkyl hydroperoxides to water and respective and Mass-Spectrometric Analysis. Cell-free extracts of M. jannaschii (25) were ox- alcohols (1). Three clustered regularly interspaced short palin- idized and treated with thiol reagents as described in SI Materials and Methods. dromic repeats (CRISPR)-associated proteins, namely Csm 2-, Methods for reducing this preparation with Trx, fluorescent labeling, and iden- 3-, and 5-family proteins, were also targeted by Trx1 (Table 1 tifying the potential Trx targets are also given in SI Materials and Methods.

Susanti et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 Activation and Activity Assay for Mtd and Fsr. Fsr was oxidized by aerobic concentration (to 100 mM, pH 7.0) and including KCl (0.5 M). KCl enhanced

dialysis and Mtd via a reaction with an oxidant, H2O2,CuCl2,orAldrithiol-2. the activities of Fsr and Mtd. Oxidized preparations were activated by anaerobic incubation in the fol- lowing mixtures: Fsr: 14 μg of partially purified enzyme in a 200-μL solution ACKNOWLEDGMENTS. This work was supported by National Science Foun- containing 50 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 20 μM dation Grant MCB 1020458 (to B.M. and B.B.B.) and National Aeronautics and Space Administration Astrobiology: Exobiology and Evolutionary Biology μ Trx1, and 1 mM DTT; Mtd: homogenous enzyme (1 M) in a solution con- Grant NNX13AI05G (to B.M.). The mass spectrometric analysis was supported taining 100 mM potassium phosphate buffer (pH 7.0), 0.5 M KCl, 5 μMTrx1, by the US Department of Agriculture Agricultural Research Service Current and 0.05 mM DTT. Fsr activity was assayed as previously (25), except 100 mM Research Information System Project 5325-43000-026-00. D.S. was partially KCl was added to the assay mixture. For Mtd, a previously described assay supported by a fellowship from the Genetics, Bioinformatics, and Computa- tional Biology Graduate Program. We thank Dr. David Grahame (Uniformed (53) was modified by replacing tetrahydromethanopterin with tetrahy- Services University of the Health Sciences) for a gift of tetrahydrosarcinapterin drosarcinapterin (a gift from Dr. D. Grahame, Uniformed Services University and Dr. William Whitman (University of Georgia) for suggesting an evolution- of the Health Sciences, Bethesda, MD), changing the phosphate buffer ary implication of our observations.

1. Meyer Y, Buchanan BB, Vignols F, Reichheld JP (2009) Thioredoxins and glutaredoxins: 28. McCollom TM, Shock EL (1997) Geochemical constraints on chemolithoautotrophic Unifying elements in redox biology. Annu Rev Genet 43:335–367. metabolism by microorganisms in seafloor hydrothermal systems. Geochim Cosmochim 2. Atkinson HJ, Babbitt PC (2009) An atlas of the thioredoxin fold class reveals the Acta 61(20):4375–4391. complexity of function-enabling adaptations. PLoS Comput Biol 5(10):e1000541. 29. Reeve JN, Nölling J, Morgan RM, Smith DR (1997) Methanogenesis: Genes, genomes, 3. Hernandez HH, Jaquez OA, Hamill MJ, Elliott SJ, Drennan CL (2008) Thioredoxin re- and who’s on first? J Bacteriol 179(19):5975–5986. ductase from Thermoplasma acidophilum: A new twist on redox regulation. Bio- 30. Mukhopadhyay B, Johnson EF, Wolfe RS (2000) A novel pH2 control on the expression chemistry 47(37):9728–9737. of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon 4. Jeon SJ, Ishikawa K (2002) Identification and characterization of thioredoxin and Methanococcus jannaschii. Proc Natl Acad Sci USA 97(21):11522–11527.

thioredoxin reductase from Aeropyrum pernix K1. Eur J Biochem 269(22):5423–5430. 31. Zirngibl C, et al. (1992) H2-forming methylenetetrahydromethanopterin dehydrogenase, 5. Kashima Y, Ishikawa K (2003) A hyperthermostable novel protein-disulfide oxidore- a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea. Eur J ductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Biochem 208(2):511–520. Pyrococcus horikoshii. Arch Biochem Biophys 418(2):179–185. 32. Alkhalfioui F, et al. (2007) Thioredoxin-linked proteins are reduced during germina- 6. Esposito L, et al. (2012) Crystallographic and spectroscopic characterizations of Sul- tion of Medicago truncatula seeds. Plant Physiol 144(3):1559–1579.

folobus solfataricus TrxA1 provide insights into the determinants of thioredoxin fold 33. Kiener A, Orme-Johnson WH, Walsh CT (1988) Reversible conversion of coenzyme F420 stability. J Struct Biol 177(2):506–512. to the 8-OH-AMP and 8-OH-GMP esters, F390-A and F390-G, on oxygen exposure and 7. Amegbey GY, Monzavi H, Habibi-Nazhad B, Bhattacharyya S, Wishart DS (2003) reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum. Arch Structural and functional characterization of a thioredoxin-like protein (Mt0807) Microbiol 150(3):249–253. from Methanobacterium thermoautotrophicum. Biochemistry 42(26):8001–8010. 34. Wedzicha BL (1992) Chemistry of sulphiting agents in food. Food Addit Contam 9(5): 8. Bhattacharyya S, et al. (2002) Identification of a novel archaebacterial thioredoxin: 449–459. Determination of function through structure. Biochemistry 41(15):4760–4770. 35. Vensel WH, et al. (2005) Developmental changes in the metabolic protein profiles of 9. Lee DY, Ahn B-Y, Kim K-S (2000) A thioredoxin from the hyperthermophilic archaeon wheat endosperm. Proteomics 5(6):1594–1611. Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities. 36. Vita N, Hatchikian EC, Nouailler M, Dolla A, Pieulle L (2008) Disulfide bond-dependent Biochemistry 39(22):6652–6659. mechanism of protection against oxidative stress in pyruvate-ferredoxin oxidore- 10. Jones WJ, Leigh JA, Mayer F, Woese CR, Wolfe RS (1983) Methanococcus jannaschii sp. ductase of anaerobic Desulfovibrio bacteria. Biochemistry 47(3):957–964. nov., an extreme thermophilic methanogen from a submarine hydrothermal vent. 37. Pieulle L, et al. (2011) Study of the thiol/disulfide redox systems of the anaerobe Arch Microbiol 136:254–261. Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target 11. Leigh JA (2002) Evolution of energy metabolism. Biodiversity of Microbial Life: for thioredoxin 1. J Biol Chem 286(10):7812–7821. Foundation of Earth Biosphere, eds Staley J, Reysenbach A (Wiley, New York), 38. Lin WC, Yang YL, Whitman WB (2003) The anabolic pyruvate oxidoreductase from pp 103–120. Methanococcus maripaludis. Arch Microbiol 179(6):444–456. 12. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein 39. DeMoll E, Grahame DA, Harnly JM, Tsai L, Stadtman TC (1987) Purification and database search programs. Nucleic Acids Res 25(17):3389–3402. properties of carbon monoxide dehydrogenase from Methanococcus vannielii. 13. Eklund H, Gleason FK, Holmgren A (1991) Structural and functional relations among J Bacteriol 169(9):3916–3920. thioredoxins of different species. Proteins 11(1):13–28. 40. de Poorter LM, Geerts WJ, Keltjens JT (2005) Hydrogen concentrations in methane- 14. Kurr M, et al. (1991) Methanopyrus kandleri, gen. and sp. nov. represents a novel forming cells probed by the ratios of reduced and oxidized coenzyme F420. Micro- group of hyperthermophilic methanogens, growing at 110°C. Arch Microbiol 156(4): biology 151(Pt 5):1697–1705. 239–247. 41. Hendrickson EL, Haydock AK, Moore BC, Whitman WB, Leigh JA (2007) Functionally 15. Cave JW, et al. (2001) Solution nuclear magnetic resonance structure of a protein distinct genes regulated by hydrogen limitation and growth rate in methanogenic disulfide oxidoreductase from Methanococcus jannaschii. Protein Sci 10(2):384–396. Archaea. Proc Natl Acad Sci USA 104(21):8930–8934. 16. Balmer Y, et al. (2003) Proteomics gives insight into the regulatory function of 42. Graham DE, Xu H, White RH (2002) Identification of coenzyme M biosynthetic chloroplast thioredoxins. Proc Natl Acad Sci USA 100(1):370–375. phosphosulfolactate synthase: A new family of sulfonate-biosynthesizing enzymes. 17. Vensel WH, Dupont FM, Sloane S, Altenbach SB (2011) Effect of cleavage enzyme, J Biol Chem 277(16):13421–13429. search algorithm and decoy database on mass spectrometric identification of wheat 43. Kumar JK, Tabor S, Richardson CC (2004) Proteomic analysis of thioredoxin-targeted gluten proteins. Phytochemistry 72(10):1154–1161. proteins in Escherichia coli. Proc Natl Acad Sci USA 101(11):3759–3764. 18. Susanti D, Mukhopadhyay B (2012) An intertwined evolutionary history of meth- 44. Löwe J, Amos LA (2000) Helical tubes of FtsZ from Methanococcus jannaschii. Biol anogenic archaea and sulfate reduction. PLoS One 7(9):e45313. Chem 381(9-10):993–999. 19. Boone DR, Whitman WB, Rouviere P (1993) Diversity and taxonomy of methanogens. 45. König H, Stetter KO (1986) Studies on archaebacterial S-layers. Syst Appl Microbiol Methanogenesis: Ecology, Physiology, Biochemistry and Genetics, ed Ferry JG (Chapman 7(2–3):300–309. and Hall, New York), pp 35–80. 46. Xia Q, et al. (2009) Quantitative proteomics of nutrient limitation in the hydro- 20. Zinder SH (1993) Physiological Ecology of Methanogens. Methanogenesis: Ecology, genotrophic methanogen Methanococcus maripaludis. BMC Microbiol 9:149. Physiology, Biochemistry and Genetics, ed Ferry JG (Chapman & Hall, New York), 47. Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune pp 128–206. systems in bacteria and archaea. Annu Rev Biochem 82:237–266. 21. Nelson-Sathi S, et al. (2012) Acquisition of 1,000 eubacterial genes physiologically 48. Zaffagnini M, et al. (2012) Glutathionylation in the photosynthetic model organism transformed a methanogen at the origin of Haloarchaea. Proc Natl Acad Sci USA Chlamydomonas reinhardtii: A proteomic survey. Mol Cell Proteomics 11(2):014142. 109(50):20537–20542. 49. Kah LC, Lyons TW, Frank TD (2004) Low marine sulphate and protracted oxygenation 22. Quan S, Schneider I, Pan J, Von Hacht A, Bardwell JC (2007) The CXXC motif is more of the Proterozoic biosphere. Nature 431(7010):834–838. than a redox rheostat. J Biol Chem 282(39):28823–28833. 50. Case CL, Rodriguez JR, Mukhopadhyay B (2009) Characterization of an NADH oxidase 23. Verdoucq L, Vignols F, Jacquot J-P, Chartier Y, Meyer Y (1999) In vivo characterization of the flavin-dependent disulfide reductase family from Methanocaldococcus jan- of a thioredoxin h target protein defines a new peroxiredoxin family. J Biol Chem naschii. Microbiology 155(Pt 1):69–79. 274(28):19714–19722. 51. Purwantini E, Mukhopadhyay B, Spencer RW, Daniels L (1992) Effect of temperature

24. Thauer RK (2012) The Wolfe cycle comes full circle. Proc Natl Acad Sci USA 109(38): on the spectral properties of coenzyme F420 and related compounds. Analytical bio- 15084–15085. chemistry 205(2):342–350. 25. Johnson EF, Mukhopadhyay B (2005) A new type of sulfite reductase, a novel co- 52. Holmgren A (1977) Bovine thioredoxin system. Purification of thioredoxin reductase

enzyme F420-dependent enzyme, from the methanarchaeon Methanocaldococcus from calf liver and thymus and studies of its function in disulfide reduction. The jannaschii. J Biol Chem 280(46):38776–38786. Journal of Biological Chemistry 252(13):4600–4606. 26. Montrichard F, et al. (2009) Thioredoxin targets in plants: The first 30 years. J Proteomics 53. Mukhopadhyay B, Daniels L (1989) Aerobic purification of N5,N10-methylenete- 72(3):452–474. trahydromethanopterin dehydrogenase, separated from N5,N10-methylenetetrahy- 27. Jannasch HW (1989) Chemosynthetically sustained ecosystems in the deep sea. dromethanopterin cyclohydrolase, from Methanobacterium thermoautotrophicum Autotrophic Bacteria, eds Schlegel HG, Bowien B (Springer, New York), pp 147–166. strain Marburg. Can J Microbiol 35(4):499–507.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1324240111 Susanti et al. Downloaded by guest on October 2, 2021