Thioredoxin targets fundamental processes in a methane

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 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.
methanogenic archaea
early Earth evolution
|
strict anaerobes that produce methane, a prominent greenhouse
gas and important fuel. We have focused on Methanocaldococcus
jannaschii—a deeply rooted, hyperthermophilic methanogen living
in deep-sea hydrothermal vents (10) where conditions mimic those
of early Earth. M. jannaschii produces methane exclusively from
H2 and CO2 via a process believed to represent an ancient form of
respiration (11). M. jannaschii thus presents an opportunity to
explore the role of Trx in an archaeon and, at the same time, gain
insight into the evolutionary history of redox regulation. Our
results suggest that Trx alleviates oxidative stress in methanogens
via a thiol-based mechanism that could also regulate fundamental
processes by redox transitions in the absence of O2. The role
formulated for this anaerobic archaeon confirms and extends
that established for aerobic forms of life.
Results
Thioredoxin Homologs of Methanarchaea. Iterative BLAST searches
(12) using Escherichia coli and M. jannaschii Trxs as queries and
screening output for hits with the C-X-X-C motif and appropriate
sizes of 70- to 110-aa residues (13) showed that Trx homologs exist
in almost all methanogen genomes represented in the National
Center for Biotechnology Information (NCBI) database (Fig. 1
and Table S1). Methanopyrus kandleri AV19, a hydrothermal
vent-associated hyperthermophilic methanogen (optimum growth
Significance
This study extends thioredoxin (Trx)-based oxidative redox
regulation to the archaea, the third domain of life. Our study
suggests that Trx is nearly ubiquitous in anaerobic methanogens, enabling them to recover from oxidative stress and
synchronize cellular processes, including methane biogenesis,
with the availability of reductants. As methane is a valuable
fuel, an end product of anaerobic biodegradation and a potent
greenhouse gas, Trx may now be considered a critical participant in the global carbon cycle, climate change, and bioenergy
production. Because methanogenesis developed before the
oxygenation of the earth, our work raises the possibility that
Trx functioned in a complex redox regulatory network in anaerobic prokaryotes at least 2.5 billion years ago.
| redox regulation | hydrothermal vent |
T
hioredoxins (Trxs) are small (∼12-kDa) redox proteins typically bearing a characteristic Cys-Gly-Pro-Cys motif that reduce specific disulfide bonds of selected proteins (1). Reduction
alters the biochemical properties of the proteins targeted—e.g., by
increasing their activity or solubility (1). Trxs are found in the three
domains of life: bacteria, eukarya, and archaea (2). In eukarya and
bacteria, the regulatory role of Trx has been shown to span the
major aspects of metabolism, including photosynthesis, biosynthesis,
replication, transcription, translation, and stress response (1). Trx
also acts as an electron donor for enzymes, notably ribonucleotide
reductase, phosphoadenosinephosphosulfate reductase, methionine
sulfoxide reductase, and peroxiredoxins (1). However, in contrast
to the wealth of information for bacteria and eukaryotes, our understanding of archaeal Trx is limited to its biochemical and structural properties (3–9). Its physiological role remains a mystery.
To help fill this gap, we have investigated the role of Trx in a
group of archaea known as methanogens or methanarchaea—
2608–2613 | PNAS | February 18, 2014 | vol. 111 | no. 7
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.,
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.
The authors declare no conflict of interest.
1
Present address: Department of Intensive Care and Intermediate Care, University Hospital,
Rheinisch-Westfaelische Technische Hochschule Aachen University, 52074 Aachen, Germany.
2
To whom correspondence may be addressed. E-mail: [email protected] or view@
berkeley.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1324240111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1324240111
Trxs of M. jannaschii. M. jannaschii (Mj) carries two Trx homologs,
Mj_0307 and Mj_0581 (9, 15), here called Trx1 and Trx2, respectively. The sequence identity and similarity between Trx1
and Trx2 are 23% and 49%, respectively. Both proteins have
homologs in Methanothermobacter thermautotrophicus ΔH (7, 8),
where Trx1 is closely related to MTH807 (identity, 51%; similarity, 67%) and Trx2 corresponds to MTH895 (identity, 37%;
similarity, 54%). Purified recombinant Trx1 and Trx2 were reduced by dithiothreitol (DTT) (Fig. S1A). However, the proteins
were distinct in two well-characterized activities in which Trx1
exhibited a closer resemblance to E. coli Trx, a standard in the field.
First, in the insulin reduction assay, Trx1 showed 80-fold higher
activity than Trx2, and Trx2 exhibited a longer lag, 35 vs. 10 min for
Trx1 (Fig. S1B). Second, Trx1 but not Trx2 was reduced by E. coli
nicotinamide adenine dinucleotide phosphate (NADP)-thioredoxin
reductase (Ec-NTR) with NADPH. It is noteworthy that the homolog of Trx2, M. thermautotrophicus ΔH (MTH895), unlike the M.
jannaschii protein, accepts electrons from E. coli NTR (8).
Identification of Trx1 Targets. A fluorescent gel/proteomics approach that proved successful in several plant investigations (1, 16)
was used to identify the M. jannaschii proteins reduced by Trx1
(Trx1 targets). Briefly, in this procedure, M. jannaschii cell extracts
were oxidized by aerobic dialysis, and the remaining free sulfhydryl
groups of the air-exposed proteins were blocked by alkylation. The
extract was then treated with Trx1 using either DTT or NADPH
(plus E. coli NTR) as reductant, anticipating that Trx1 would reduce the regulatory disulfide (S–S) groups formed in aerobic dialysis. The newly available free –SH groups were derivatized with the
fluorescent probe monobromobimane (mBBr), and the labeled
proteins were resolved in 2D gels (Fig. S2 A and B). The fluorescent
spots, which were either absent or less intense in control gels, were
analyzed by mass spectrometry (17). The experiment with DTT was
performed in triplicate and that with Ec-NTR+NAPDH was performed once. From these experiments, we identified a total of 152
potential Trx1 targets (Table 1 and Table S2). Of these, 19 proteins
were identified in all four experiments, and 18, 38, and 77 were
detected in three, two, and one of the experiments, respectively.
Effect of Reduction by Trx1 on the Activity of Selected M. jannaschii
Enzymes. F420-dependent sulfite reductase. An air-exposed 7,8-dide-
methyl-8-hydroxy-5-deazaflavin-5′-phosphoryllactyl glutamate [coenzyme F420 (F420)]-dependent sulfite reductase (Fsr) preparation
showed two-thirds the activity observed with the corresponding
anaerobic preparation, the respective values being 0.132 and 0.200
U/mg. Assay of oxidized Fsr with Trx1 (20 μM) at 65 °C in the
presence of 1 mM DTT increased the activity of the enzyme by 2.9fold (Fig. 2); with twice as much Trx1, activation was 4.6-fold. DTT
alone (1 mM) inhibited the enzyme, and Trx1 alone activated Fsr
1.5-fold, likely due to a protein concentration effect.
F420-dependent methylenetetrahydromethanopterin dehydrogenase. Nonreducing SDS/PAGE developed with a monobromobimane (mBBr)treated preparation revealed that purified recombinant F420dependent 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, confidence values ≥700 (out of 1,000 replicates). Scale
bar, number of base substitution per site. The 16SrRNA 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.
Susanti et al.
PNAS | February 18, 2014 | vol. 111 | no. 7 | 2609
ECOLOGY
temperature, 98 °C), was apparently the only exception in lacking a
recognizable homolog of Trx (14).
Methanococci and Methanobacteria carried an average of two Trx
homologs, with their numbers ranging from one to four, whereas
Methanomicrobia possessed two to eight Trx homologs, with an
average of four. Methanocorpuscullum labreanum, a member of the
latter class, was an exception in possessing two Trx homologs.
Table 1. Potential M. jannaschii Trx1 targets
Metabolic function or
structural unit
ATP synthesis
Biosynthesis
Coenzyme M biosynthesis
Defense against
foreign DNA
Hypothetical protein
Metabolism
Methanogenesis
(energy generation)
Miscellaneous
Nitrogen and amino
acid metabolism
Oxidative stress response
Replication, transcription,
and translation
Structural proteins
Sulfite detoxification
Transport proteins
Potential targets
V-type ATP synthase subunit A*, V-type ATP synthase subunit B*
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*
Phosphosulfolactate synthase
Csm3 family CRISPR-associated RAMP protein
Hypothetical protein MJ_0164, hypothetical protein MJ_0308, hypothetical protein MJ_1099
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*
F420-dependent methylenetetrahydromethanopterin dehydrogenase,
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
AMMECR 1 domain protein, methanogenesis marker protein 17, methyltransferase,
iron-sulfur flavoprotein
(R)-2-Hydroxyglutaryl-CoA dehydratase activator, 2-hydroxyglutaryl-CoA dehydratase,
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*
Flavoprotein FpaA, NADH oxidase, peroxiredoxin*
30S ribosomal protein S7, 50S ribosomal protein L6, acidic ribosomal protein P0, arginyl-tRNA
synthetase, cell division protein CDC48*, cell division protein FtsZ I*, elongation factor 1-α*,
elongation factor EF-2, thermosome
Flagella-like protein E, S-layer protein
F420-dependent sulfite reductase†
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 potential redox active cystine disulfides, the enzyme was treated
with several oxidants, H2O2, CuCl2, and Aldrithiol-2. Based on
mBBr gel analysis, Aldrithiol-2 proved most effective in oxidizing
Mtd. This oxidation deactivated the enzyme by 53%. A treatment of the deactivated enzyme with fivefold molar excess Trx1
and 0.05 mM DTT yielded a 4.4-fold increase in activity vs. a 1.4fold enhancement seen with DTT alone (Fig. 2).
Discussion
Distribution of Trx Homologs in Methanogens. The evidence presented above suggests that Trx homologs are nearly universal in
methanogenic archaea. M. kandleri, the most deeply rooted and
the most thermophilic methanogen known (growth occurs at
84–110 °C), was the only exception. This organism, which is
solely dependent on H2 and CO2 for methanogenesis (14),
lacked a recognizable homolog of Trx. In the other methanogens, the distribution of Trx homologs followed a pattern (Fig.
1 and Table S1). Phylogenetically deeply rooted representatives
belonging to the classes of Methanococci and Methanobacteria
2610 | www.pnas.org/cgi/doi/10.1073/pnas.1324240111
carried a limited number of Trx homologs (two to four; two on
average). These organisms have relatively smaller genomes (1.24–
2.94 Mbp; NCBI data), include almost all hyperthermophilic or
thermophilic methanogens (19), and are mostly restricted to H2dependent methanogenesis (19). By contrast, the late-evolving
Methanomicrobia with larger genomes (1.8–5.75 Mbp; NCBI
data) and more complex metabolism carried up to eight Trxs (on
average, four). The methylotrophic Methanomicrobia use methanol and methylamines, and some of these perform methanogenesis from acetate as well as H2 and CO2 (19, 20); most are
mesophiles and relatively O2 tolerant.
It is possible that the Trx system came into play in the deeply
rooted methanogenic archaea as these organisms faced a more
oxidizing environment brought about by H2 limitation or O2
exposure. It remains to be seen whether the larger number of Trxs
in late-evolving methanogens is a result of horizontal gene transfer
or gene duplication coupled with subsequent diversification. We
note that these organisms’ ability to use a range of methanogenic
substrates is thought to be due to a large number of genes acquired from the Clostridia and other anaerobes (21).
Susanti et al.
M. jannaschii Trxs. Trx1 and Trx2 were distinct in terms of amino
acid sequence, reactivity with insulin, and activity with E. coli
NTR. These features are possibly related to the nature of the
putative redox active site motif C-X-X-C as the two internal
residues (X’s) influence the redox properties of the protein (22).
In Trx1 and Trx2, this motif is, respectively, C-P-H-C and C-P-K-C,
which differ from each other and from the classical C-G-P-C (21). It
is thus not surprising that Trx1 and Trx2 showed different specificities. Trx1 was typical—i.e., similar to its E. coli counterpart in
primary structure, robust insulin reduction activity, and reduction
by E. coli NTR. This protein was, therefore, chosen for identifying
candidate Trx target proteins in M. jannaschii.
Targets of Trx1. Proteomics and enzyme activity measurements
suggested that Trx1 influences multiple processes in M. jannaschii,
including methanogenesis—the hallmark of the methanogens. Our
proteomics analysis revealed a total of 152 M. jannaschii polypeptides as potential Trx1 targets, representing ∼10% of the total
ORFs in the organism’s genome (Table S2). Of these, 75 targets
were detected in at least two of four independent experiments
(Table 1) and more than one-half were observed only once. As
shown in Table S2, most of the targets contain at least two Cys
residues, indicative of Trx-reducible intramolecular or intermolecular Cys disulfide bonds (Table S2). Curiously, a few of the
targets have only one Cys, raising the possibility that in these
instances Trx reduces intermolecular disulfides as described for
yeast 1-Cys peroxiredoxin (23). The putative peroxiredoxin of M.
jannaschii (MJ_0736), however, contains five Cys.
The Trx1 target proteins participate in multiple processes in
addition to methanogenesis: biosynthesis, information processing, cell division, sulfite detoxification, oxidative response, and
resistance to phages and invasion by foreign DNA. Structural
proteins were also identified as Trx1 targets. The results reveal
the obvious vulnerability of an ancient methanogen cell to oxidative
stress and the suitability of Trx for repairing the resulting damage.
To confirm and extend the proteomics results, we tested the effect of reduced Trx1 on the in vitro activity of two candidate target
enzymes: Mtd, a core enzyme of the methanogenesis pathway (24),
and Fsr (25), an enzyme that enables certain methanogens to tolerate and use sulfite as a source of sulfur. Oxidized forms of both
enzymes were activated by Trx1, giving further credence to the
fluorescent/gel approach of target identification. Mtd and Fsr were
selected based on two criteria: being specific to methanarchaea and
the availability of assay tools in our laboratories.
M. jannaschii Systems Targeted by Trx1. Methanogenesis. It is significant that many of the enzymes identified as Trx targets function in
the reduction of CO2 to CH4 (Fig. 3, Table 1, and Table S2).
Based on the nature of our experiments, this observation suggests
that, reminiscent of plants, Trx activates enzymes of M. jannaschii
that have been deactivated following O2 exposure (1, 26). The
Susanti et al.
PNAS | February 18, 2014 | vol. 111 | no. 7 | 2611
ECOLOGY
Fig. 2. Activation of F420 -dependent sulfite reductase or Fsr and F 420 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.
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 anaerobic conditions. In the deep-sea hydrothermal vents M. jannaschii 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 transcriptionally regulated by H2 availability, and our observations suggest
the presence of parallel posttranslational control effected by Trx
(Table 1 and Table S2; Fig. 2). This possibility is also consistent
with the proposal that Mtd is the primary enzyme for the reduction of methylenetetrahydromethanopterin under H2 limitation (29, 30) and with our finding of a direct effect of Trx on the
activity of the enzyme. Significantly, the alternate H2-dependent
enzyme [H2-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd)], also a potential Trx1 target, is active under
high H2 partial pressure (31). In view of these results, it is timely
to determine whether Trx regulates the activity of Hmd and
other methanogenesis enzymes identified as targets (Table 1 and
Table S2) as well as counterparts in terrestrial systems where O2
exposure and changes in reductant supply are common (19, 20).
The ability to down-regulate methanogenesis, the sole avenue for
energy generation, via the oxidation of sulfhydryl groups would
enable methanogens to attain a dormant-type state. With the return
of favorable environmental conditions, Trx could reactivate target
methanogenesis enzymes via disulfide reduction and thereby restore
growth and other vital cell processes. This situation resembles the
role of Trx in initiating processes associated with seed germination
(32). Modification of F420 via adenylation and guanylation provides
an alternate avenue for enabling a methanogen to shut down energy
production and achieve a dormant state (33).
Sulfite detoxification. Identified as a target in proteomics studies
(Table S2), Fsr was deactivated upon exposure to O2, and the
altered enzyme was partially reactivated by reduced Trx1 (Fig. 2).
These observations make physiological sense. Sulfite inhibits
methyl-coenzyme M reductase and, thereby, impedes methanogenesis (25). In the habitat of M. jannaschii, sulfite is formed when
O2-containing cold seawater mixes with the hot sulfide-rich vent
fluid (25). Fsr detoxifies the newly formed sulfite by reducing it to
sulfide, an essential nutrient for methanogens (25). Because sulfite can oxidize protein sulfhydryl (SH) groups to the disulfide
(S–S) level (34), it is not surprising that oxidatively deactivated Fsr
can be reductively activated by Trx1. It is possible that activation by
Trx is a general feature of sulfite reductases as the enzyme of wheat
starchy endosperm appears also to be a Trx target (35).
Biosynthesis. The de novo synthesis of acetate and pyruvate from
CO2 are key initial anabolic steps for an autotroph such as
M. jannaschii (10). It is significant that the two enzymes of this
process, acetyl-CoA decarbonylase/synthase (ACDS) and pyruvate:ferredoxin oxidoreductase (PFOR), were both identified as
Trx1 targets (Table 1 and Fig. 3). Trx is known to revive oxidatively damaged PFOR in Desulfovibrio africanus, an anaerobic
sulfate reducing bacterium (36, 37). However, the situation may be
different in M. jannaschii as PFOR and ACDS of methanogens
have been reported to be irreversibly inactivated by O2 exposure in
vitro (38, 39). In methanogens, the role of Trx could lie in recovery
from less severe O2 exposure or in redox regulation of activity.
Methanogens could invoke the latter in response to a drop in H2
partial pressure—a fact of life in most, if not all, of their natural
habitats (19, 20, 25, 27, 28). In these cases, the prevailing midpoint
potential values of H+/H2 redox couple would make the formation
of protein disulfides thermodynamically feasible. Because the level
of coenzyme F420 in a methanogen cell is in equilibrium with
environmental H2 partial pressure (40, 41), this mechanism of
sulfhydryl oxidation could be performed by an F420-dependent
enzyme acting directly or via an intermediary.
The synthesis of sugars via gluconeogenesis is an energyintensive pathway that requires a reductant. In eukarya, many of
the associated enzymes are linked to Trx (26). Significantly,
Fig. 3. Select reactions and pathways of M. jannaschii
targeted by Trx1 (Mj_0307). The methanogenesis pathway 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, 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-H4MPTcoenzyme 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.
several of their M. jannaschii counterparts were also identified as
potential Trx targets (Fig. 3; Table 1 and Table S2), suggesting
that gluconeogenesis could also be redox regulated in this organism. Following this same theme, several enzymes of amino
acid biosynthesis reported to be Trx targets in plants (26)—
notably, glutamine synthetase, threonine synthase, and aspartate
semialdehyde dehydrogenase were reduced by Trx1 in M. jannaschii
extracts. Phosphosulfolactate synthase, an enzyme needed for the
biosynthesis of coenzyme M—a requirement for methane formation
with all substrates (42), also appeared to be linked to Trx1 (42). A
role in coenzyme M synthesis falls within the broader function of
Trx in the repair and regulation of the methanogenesis process.
Transcription, translation, and cell division. Transcription and translation have previously been linked to Trx-based regulation in
Bacteria and Eukarya (1). Modification of the RNA polymerase
ω subunit and elongation factors in E. coli (43) and several
chloroplast ribosomal proteins fall in this category (26). Similar
controls likely exist in M. jannaschii where a ribosomal protein S7
and several tRNA synthetases were identified as Trx1 targets
(Table 1 and Table S2).
Like its counterparts in chloroplasts and E. coli (16, 43), FtsZ,
a cytoskeletal protein similar to tubulin in eukaryotes (44), was
reduced by Trx1 in M. jannaschii (Table 1 and Table S2). Thus, as
with E. coli, Trx acting through FtsZ could contribute to the
regulation of cell division in this organism.
Structural proteins. One of the two S-layer proteins, which are
major cell envelope components in methanogens (45), was reduced by Trx1. Interestingly, the level of S-layer protein decreases
under H2 limitation in Methanococcus maripaludis, a close relative
of M. jannaschii (46). Considering that Trx is a posttranslational
modifier, it is possible that both the generation and assembly of
the S-layer is redox controlled in methanogens.
Defense against reactive oxygen species and foreign DNA. Similar to
chloroplasts (16), a peroxiredoxin was identified as a Trx target
in M. jannaschii (Table 1 and Table S2). Peroxiredoxins are
critical antioxidant enzymes catalyzing the reduction of hydroperoxides and alkyl hydroperoxides to water and respective
alcohols (1). Three clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, namely Csm 2-,
3-, and 5-family proteins, were also targeted by Trx1 (Table 1
2612 | www.pnas.org/cgi/doi/10.1073/pnas.1324240111
and Table S2). CRISPR elements and associated proteins provide defense against invasion by external DNA materials such
as plasmids and phage in archaea as well as bacteria (47),
raising the possibility that this process is regulated by Trx in
M. jannaschii.
Concluding Remarks
The present work extends Trx-based redox transition to the third
domain of life. The methanarchaeon M. jannaschii was found to
use this protein to protect a range of cellular processes against
oxidative damage. Interestingly, many of the Trx targets identified have counterparts in plants where an oxidative type of regulation is known to occur (16, 48).
The present findings have far-reaching implications to our
understanding of the evolution of redox regulation as well as
to areas of current societal interest. Because M. jannaschii
performs hydrogenotrophic methanogenesis, a process that developed before the appearance of O2 (11, 49), Trx may have
originally functioned in an anaerobic regulatory capacity in this
ancient organism. Its participation in protecting cells against O2
would have developed later. Accordingly, the redox network
created by Trx, and the attendant cellular complexity, would have
developed in prokaryotes at least 2.5 billion years ago. Future
research will be directed toward this question. On the pragmatic
side, due to the role of methanogens in producing methane and
the attendant changes in the biosphere, Trx emerges as a key
participant in the global carbon cycle, climate change, and
bioenergy production.
Materials and Methods
Purified Preparations of M. jannaschii Trxs, Mtd, and Fsr, and Methanogen
Cofactors, and Insulin Reduction Assay. Previously described methods were
used for generating homogeneous preparations of recombinant His-tagged
Trx and Mtd (50) and F420 (51), partial purification of Fsr from M. jannaschii
cell extracts (25), and the insulin assay for Trx (52).
Trx-Mediated Reduction of M. jannaschii Cell Extract Proteins, 2D Gel Electrophoresis,
and Mass-Spectrometric Analysis. Cell-free extracts of M. jannaschii (25) were oxidized and treated with thiol reagents as described in SI Materials and Methods.
Methods for reducing this preparation with Trx, fluorescent labeling, and identifying the potential Trx targets are also given in SI Materials and Methods.
Susanti et al.
Activation and Activity Assay for Mtd and Fsr. Fsr was oxidized by aerobic
dialysis and Mtd via a reaction with an oxidant, H2O2, CuCl2, or Aldrithiol-2.
Oxidized preparations were activated by anaerobic incubation in the following mixtures: Fsr: 14 μg of partially purified enzyme in a 200-μL solution
containing 50 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 20 μM
Trx1, and 1 mM DTT; Mtd: homogenous enzyme (1 μM) in a solution containing 100 mM potassium phosphate buffer (pH 7.0), 0.5 M KCl, 5 μM Trx1,
and 0.05 mM DTT. Fsr activity was assayed as previously (25), except 100 mM
KCl was added to the assay mixture. For Mtd, a previously described assay
(53) was modified by replacing tetrahydromethanopterin with tetrahydrosarcinapterin (a gift from Dr. D. Grahame, Uniformed Services University
of the Health Sciences, Bethesda, MD), changing the phosphate buffer
concentration (to 100 mM, pH 7.0) and including KCl (0.5 M). KCl enhanced
the activities of Fsr and Mtd.
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Susanti et al.
PNAS | February 18, 2014 | vol. 111 | no. 7 | 2613
ECOLOGY
ACKNOWLEDGMENTS. This work was supported by National Science Foundation Grant MCB 1020458 (to B.M. and B.B.B.) and National Aeronautics and
Space Administration Astrobiology: Exobiology and Evolutionary Biology
Grant NNX13AI05G (to B.M.). The mass spectrometric analysis was supported
by the US Department of Agriculture Agricultural Research Service Current
Research Information System Project 5325-43000-026-00. D.S. was partially
supported by a fellowship from the Genetics, Bioinformatics, and Computational Biology Graduate Program. We thank Dr. David Grahame (Uniformed
Services University of the Health Sciences) for a gift of tetrahydrosarcinapterin
and Dr. William Whitman (University of Georgia) for suggesting an evolutionary implication of our observations.

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