Structural and Biochemical Characterizations of Methanoredoxin from

Dec 18, 2015 - Glutaredoxins (GRXs) are thiol–disulfide oxidoreductases abundant in prokaryotes, although little is understood of these enzymes from...
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Structural and Biochemical Characterizations of Methanoredoxin from Methanosarcina acetivorans, a Glutaredoxin-Like Enzyme with Coenzyme M‑Dependent Protein Disulfide Reductase Activity Deepa Yenugudhati,†,∥ Divya Prakash,†,∥ Adepu K. Kumar,†,§ R. Siva Sai Kumar,† Neela H. Yennawar,*,‡ Hemant P. Yennawar,*,† and James G. Ferry*,† †

Department of Biochemistry and Molecular Biology, ‡Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: Glutaredoxins (GRXs) are thiol−disulfide oxidoreductases abundant in prokaryotes, although little is understood of these enzymes from the domain Archaea. The numerous characterized GRXs from the domain Bacteria utilize a diversity of lowmolecular-weight thiols in addition to glutathione as reductants. We report here the biochemical and structural properties of a GRX-like protein named methanoredoxin (MRX) from Methanosarcina acetivorans of the domain Archaea. MRX utilizes coenzyme M (CoMSH) as reductant for insulin disulfide reductase activity, which adds to the diversity of thiol protectants in prokaryotes. Cell-free extracts of M. acetivorans displayed CoMS-SCoM reductase activity that complements the CoMSHdependent activity of MRX. The crystal structure exhibits a classic thioredoxinglutaredoxin fold comprising three α-helices surrounding four antiparallel β-sheets. A pocket on the surface contains a CVWC motif, identifying the active site with architecture similar to GRXs. Although it is a monomer in solution, the crystal lattice has four monomers in a dimer of dimers arrangement. A cadmium ion is found within the active site of each monomer. Two such ions stabilize the N-terminal tails and dimer interfaces. Our modeling studies indicate that CoMSH and glutathione (GSH) bind to the active site of MRX similar to the binding of GSH in GRXs, although there are differences in the amino acid composition of the binding motifs. The results, combined with our bioinformatic analyses, show that MRX represents a class of GRX-like enzymes present in a diversity of methane-producing Archaea.

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sequenced genomes of species belonging to all domains of life (Archaea, Bacteria, and Eukarya) are annotated for dithiol GRX family enzymes which contain a consensus C[P/S][Y/F]C active-site motif. The monothiol GRX family contains a CGFSlike active-site sequence and is reported to be present only in the domains Bacteria and Eukarya.6 The third family is dominant in plants with the consensus active-site sequence CC[M/L][C/S]. Annotations of genomic sequences indicate that TRXs and GRXs are widely represented in the domain Archaea (NCBI database). Although a few TRXs have been investigated,7−14 the only GRX characterized from the domain Archaea is that from the nonmethanogen Pyrococcus furiosus.15 Here, we describe the biochemical and structural properties of a dithiol GRX-like enzyme from the methane-producing archaeon Methanosarcina acetivorans that we name methanoredoxin (MRX). Notably, coenzyme M (−O3SCH2CH2SH) is a reductant for MRX, which adds to the list of low-molecularweight thiol protective systems in prokaryotes and defines a

lutaredoxin (GRX) and thioredoxin (TRX) are ubiquitous redox enzymes of fundamental importance in metabolically and phylogenetically diverse organisms. Furthermore, they are ancient enzymes, providing a window into evolution.1,2 GRX and TRX systems are involved in a growing list of processes, including the reductant for enzymes such as ribonucleotide reductase and the primary means by which cysteine residues of proteins are maintained in the reduced state during oxidative stress.3−5 TRXs and GRXs also participate in redox sensing and regulation of gene expression. Iron assimilation and iron−sulfur cluster synthesis are additional functions ascribed to GRXs.3−5 Another unique function of GRXs is to catalyze glutathionylation and deglutathionylation of mammalian proteins, thereby regulating enzyme activity and protecting the target thiol group during oxidative stress. TRXs and GRXs are grouped in the thioredoxin superfamily that shares a common structural fold composed of either a fouror five-stranded β-sheet flanked by several α-helices on either side of the β-sheet. Although GRXs and TRXs are similar in structure, sequence comparisons show little similarity and the two proteins are reduced differently. GRXs are reduced nonenzymatically with reduced glutathione (GSH), whereas TRXs are reduced enzymatically by TRX reductases. Three GRX families are proposed based on active-site motifs.1 The © XXXX American Chemical Society

Received: July 22, 2015 Revised: December 16, 2015

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DOI: 10.1021/acs.biochem.5b00823 Biochemistry XXXX, XXX, XXX−XXX

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treated with cold acetone and centrifuged to remove protein, and the supernatant solution was collected for analysis. Deglutathionylation Activity. The assay measures deglutathionylation of the mixed disulfide formed by spontaneous reaction of GHS with β-hydroxyethyl disulfide (HED). The activity was performed as described.21 A mixture of 1 mM GSH, 0.4 mM NADPH, and 6 μg/mL yeast glutathione reductase was prepared in 50 mM Tris-HCl (pH 6.8). To 500 μL of this mixture was added HED at a final concentration of 0.7 mM. After 3 min of incubation, varying concentrations of MRX were added to the sample cuvette, and an equal volume of buffer only was added to the reference cuvette. The oxidation of NADPH was followed spectrophotometrically at 340 nm. Dehydroascorbate Reductase Activity. The activity was assayed as described.21 The reaction mixture contained 50 mM Tris-HCl (pH 6.8), 2 mM EDTA, 2 mM GSH, 0.1 mg/mL bovine serum albumin, 6 μg/mL yeast glutathione reductase, 200 μM NADPH, and 1 mM dehydroascorbate. After preincubating the reaction mixture for 3 min, varying concentrations of MRX were added to the cuvette and the oxidation of NADPH was followed spectrophotometrically at 340 nm. CoMS-SCoM Reductase Activity. The activity was measured spectrophotometrically by monitoring production of CoMSH with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB).19 The assay was performed anaerobically in a stoppered 1.5 mL serum vial containing 1 atm of N2 and incubated at 50 °C. The reaction mixture (0.3 mL) contained 1.2 μmol of CoMS-SCoM and 0.3 mg of cell-free extract protein from acetate-grown M. acetivorans. The reaction was initiated by the addition of 1.0 mL of CO to the headspace of the reaction vial. Aliquots of the reaction mixture were collected at intervals and assayed for free thiols using DTNB. Control experiments were performed separately in which each component of the reaction mixture was individually eliminated. A unit of enzyme activity is defined as 1.0 μmol of CoMS-SCoM reduced per min. Midpoint Potential. The relative percent of disulfide bond formation at various redox potentials was determined by assaying the fluorescence emission of MRX when reacted with monobromobimane (mBBr) as described.22,23 Prior to redox titration, oxidized MRX was prepared by incubation with a 10fold excess of potassium ferricyanide for 2 h that was removed by size-exclusion chromatography. A series of reactions were set up anaerobically at different redox levels by varying the concentration ratio of oxidized (GSSG) and reduced (GSH) glutathione at a total concentration of 5 mM in 100 mM HEPES (pH 7.0) containing 20 μM of oxidized MRX in a 400 μL reaction mixture. After 2 h of incubation, 50 μL of mBBr was added (4 mM final concentration) and the reaction was carried out in the dark for 45 min. The reaction mixture was then precipitated with 100 μL of 100% trichloroacetic acid (TCA) on ice for 30 min. The protein was pelleted by centrifugation, washed twice with 200 μL of 100% acetone, and then suspended in 1 mL of 100 mM Tris-HCl (pH 8.0) containing 1% SDS. The fluorescence emission of the MRXmBBr preparation was measured at 472 nm after excitation at 380 nm using a Hitachi F-2000 fluorescence spectrophotometer. The maximum fluorescence corresponded to the most reduced sample. The values were transformed into percentages of reduced protein and fitted to a two-electron Nernst equation using nonlinear regression for the Em value determination with OriginPro (Northampton, MA) software.

novel role for this cofactor that is central to all pathways of methanogenesis.16

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MATERIALS AND METHODS Materials. All chemicals were purchased from Sigma. The homodisulfide of CoMSH (CoMS-SCoM) was prepared as described previously.17 Analytical. Protein concentrations were determined by Bradford’s method using bovine serum albumin as standard.18 The native molecular mass of MRX was determined using a Sephadex G-50 gel filtration column equilibrated with buffer A containing 0.1 M NaCl and developed at a flow rate of 0.2 mL/ min. Dynamic light scattering analysis was performed using a Viscotek 802 instrument (Malvern Instruments) with the associated OmniSIZE software at a protein concentration of 2 mg/mL. As-purified MRX was in the reduced form, as determined by measuring free thiols with Ellman’s reagent before and after oxidation with potassium ferricyanide that was removed by size-exclusion chromatography.19 Mass spectrometric analysis of purified MRX was performed at the Penn State University Proteomics and Mass Spectrometry Core Facility. Cloning and Expression. The gene encoding MRX in the locus MA1658 was amplified from M. acetivorans genomic DNA by PCR using a forward primer (5′-TTTTTTCATATGCATCATCACCATCACCACAATATATTTAGTAAGGACA-3′) containing a NdeI recognition site and a reverse primer (5′-TTTTTTAAGCTTTTAAAAGCCAAGAGC TTCACGGATTT-3′) containing a HindIII recognition site. The underlined region in the forward primer sequence encodes six His residues. The PCR product was digested with NdeI and HindIII and cloned into the pET22b(+) vector (Novagen) to yield pMA1658. This plasmid was transformed into Escherichia coli strain Rosetta (DE3) pLacI (Novagen), which was used to inoculate Luria−Bertani broth containing 100 μg/mL ampicillin and 35 μg/mL chloramphenicol. Cells were grown at 37 °C to an A600 of 0.6−0.8 and induced with 1 mM isopropyl thiogalactopyranoside (IPTG). Then, the cultures were allowed to continue growing for 5−6 h. Cells were harvested by centrifugation and stored frozen at −80 °C until use. Enzyme Purification. Cell paste (5 g wet weight) was thawed in 30 mL of buffer A (50 mM MOPS, pH 6.8) and passed twice through a chilled French pressure cell at 138 MPa. The cell debris was removed by centrifuging the cell lysate at 29 000g for 30 min. The supernatant solution was equilibrated with buffer A and passed through a 0.45 μm filter before loading on a Ni Sepharose column pre-equilibrated with buffer A containing 10 mM imidazole. The column was washed with the same buffer to eliminate unbound protein. MRX was eluted from the column with a linear gradient of 0.01−0.25 M imidazole in buffer A. Fractions containing MRX were pooled and dialyzed against buffer A to remove salt. Insulin Disulfide Reduction Activity. The rate of insulin disulfide reduction was determined by monitoring turbidity at 650 nm as described previously except that it was performed anaerobically.20 The reaction was initiated by adding different concentrations of reductant. The activity (Vmax/τ) was calculated as described, where Vmax is the rate (ΔA650/min) determined by a line drawn through the linear portion of the curve and τ is the lag time determined by the intersection of the line with the horizontal axis.20 The reaction was monitored by the disappearance of CoMSH, CoBSH, or CoASH determined by measuring free thiols using Ellman’s reagent.19 Samples were B

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Biochemistry Crystallization. Robotic screening was done using the Phoenix crystallization robot and the Qiagen classics suite crystallization screen (www.qiagen.com). Concentrations of 65−70 mg/mL of MRX in 10 mM MOPS buffer (pH 6.8) was used. Drops were in the sitting drop geometry and incubated at 21 °C. A volume of 0.7 μL each of protein and screen solution was used for setting the sitting drops. In 2 weeks, two crystal forms, plate-like and rhomboidal, were identified. Plate-like crystals were further grown in size by the microseeding method in 0.2 M ammonium sulfate, 0.1 M sodium cacodylatetrihydrate (pH 6.5), and 30% PEG 8000. For cryofreezing of this crystal form, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate trihydrate (pH 6.5), and 50% PEG 8000 was found to be optimal. The rhomboid crystals grew in 0.1 M HEPES buffer, (pH 7.5), 0.05 M cadmium sulfate, and 1 M sodium acetate and were cryofrozen in 0.1 M HEPES buffer (pH 7.5), 0.05 M cadmium sulfate, and 2 M sodium acetate. Both crystal forms were tested for X-ray diffraction. The plate-like crystal was in the triclinic space group P1 with cell dimensions a = 69.1 Å, b = 98.2 Å, c = 110.2 Å, α = 99.8°, β = 83.0°, and γ = 105.6° and diffracted only to 3.1 Å. Furthermore, Matthews coefficient (Vm) calculations predicted 16 protein molecules in the asymmetric unit, so this crystal data was not pursued for structure solution. The rhomboid crystals belonged to tetragonal system space group P41 with unit cell dimensions a = b = 59.186 Å and c = 93.18 Å and diffracted to 2.45 Å. The crystals were soaked in 40 mM CoMSH in the crystallization mother liquor in an attempt to incorporate CoMSH in the crystal lattice. X-ray data sets on both crystal forms, triclinic and tetragonal, were collected on a Rigaku MicroMax 007 diffractometer equipped with a Saturn 944+ CCD area detector and a X-stream 2000 cryocooling device. The crystal and data collection statistics for the tetragonal form are listed in Table S1. Crystal Structure Solution. The crystal structure of the tetragonal crystal form was solved by the molecular replacement method using a monomer from the dimer structure of the Methanosarcina mazei glutaredoxin (PDB code 3NZN).24 The initial molecular replacement search yielded a dimer, and a subsequent search using the dimer lead to the tetramer structure. The structure solution and refinement were completed using the Autobuild and refinement features of the PHENIX software suite.25 This was alternated with manual electron-density model fitting using Coot software.26 MacPyMol software27 was used for model visualization and publication figures. A total of 3216 non-hydrogen atoms were refined in the asymmetric unit, which includes 119 solvent molecules and 12 cadmium atoms. Stereochemical analysis of the protein backbone shows that 96% of residues are in the allowed and favored regions of the Ramachandran map. The final R-factors, R-work = 0.2163 and R-free = 0.2842, are in the typical range for structures within this data resolution. The electron density for the 15 residues at the N-terminus is weak compared to that for the remainder of the protein. Most of the outliers in the Ramachandran map are in this region, wherein two cadmium atoms have also been identified. An attempt to soak-in CoMSH in the rhomboid crystals proved to be futile. The crystal structure shows a cadmium ion occupying the active site and blocking the substrate from binding. The crystal structure of MRX was deposited in the Protein Data Bank (PDB) as entry 5CAX. GSH and CoMSH Modeling. The solved X-ray crystal structure was the starting point to model substrates GSH and

CoMSH into the MRX active site. The substrates were oriented and positioned similar to that seen in the recently solved structures of the GSH (PDB code 4TR1) and GSSG (PDB code 4TR0) bound forms of glutaredoxin from Clostridium oremlandii. Hydrogen bonds were optimized in the MRX structure. The modeling was done in the Coot software and energy minimized using the online YASARA server.28 The models were further analyzed using PyMOL.

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RESULTS Purification and Biochemical Characterization. MRX encoded in the MA1658 locus was heterologously expressed and purified to homogeneity, as judged by SDS-PAGE (Figure S1), which also indicated a subunit molecular mass consistent with the calculated value of 11.5 kDa. Mass spectrometry analysis confirmed the identity and molecular mass. A native molecular mass of 12 kDa was estimated by gel filtration chromatography and DLS, a result indicating that the native enzyme is a monomer, which is typical of GRXs from the domain Bacteria. Purified MRX exhibited GSH-dependent insulin disulfide reductase activity (Figure 1A). However, the genome of M. acetivorans has no annotations for genes encoding enzymes catalyzing the synthesis of glutathione (NCBI database), and reports conclude that methanogens lack glutathione.29,30

Figure 1. Insulin disulfide reduction activity of M. acetivorans MRX. (A) CoMSH- and GSH-dependent activities. The reaction was initiated by the addition of 0.7 mM (final concentrations) reductants to the reaction mixture. Symbols: (●) GSH and (■) CoMSH. The GSH- and CoMSH-dependent activities were 16 and 14 (Vmax/τ, 10−4), respectively. (B) CoMSH-dependent activity coincident with disappearance of CoMSH. The reaction was initiated by the addition of 1.0 mM (final concentration) CoMSH. Symbols: (●) CoMSH consumed and (○) insulin disulfide reduction. The activity was 11.0 (Vmax/τ, 10−4). No detectable activity was observed in the absence of GRX or MRX. C

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methanogen Methanothermobacter thermautotrophicus has been purified and characterized, which prompted an examination of CoMS-SCoM reductase activity in cell-free extracts of M. acetivorans.17 The assay utilized CO to supply reductant to CO dehydrogenase that is inherent in the extract and utilizes ferredoxin as the electron acceptor.36 The extract showed COdependent CoMS-SCoM reductase activity of 0.15 ± 0.02 μmol/min/mg protein, which is substantially greater than the NADPH-dependent activity of 0.010 μmol/min/mg cell-free extract protein reported for M. thermautotrophicus.17 Although purification of the reductase requires identification of the immediate electron donor, the results establish the presence of a CoMS-SCoM reductase for which reduction is linked to the oxidation of ferredoxin. Overall Crystal Structure. The tetrameric structure of MRX has four independent monomers of 96 residues each in the asymmetric unit (Figure 3). The backbone structure of each

Coenzyme M (CoMSH) is a methyl carrier that functions in the final step of all methanogenic pathways and is hypothesized to be a redox regulator analogous to glutathione.17 Indeed, MRX showed CoMSH-dependent insulin disulfide reductase activity (Figure 1A) coincident with the disappearance of CoMSH (Figure 1B). The mean activity of three independent assays was 9.6 ± 0.5 (Vmax/τ × 10−4), and the Km value for CoMSH was 2.2 ± 0.2 mM (Figure S2A). MRX also showed GSH-dependent activity (Figure 1A), although the Km value (8.9 ± 1.3 mM) (Figure S2B) was 3-fold greater than that for CoMSH. Coenzyme B (CoBSH) is an electron donor that functions in the final step of all methanogenic pathways. No insulin disulfide reductase activity was detected when CoBSH was substituted for CoMSH. Coenzyme A (CoASH) functions exclusively in the pathway of acetate conversion to methane by all Methanosarcina species and the biosynthesis of cell carbon in all other methanogens. CoASH substituted for CoMSH in the insulin disulfide reductase assay, although the activity 6.6 ± 0.5 (Vmax/τ × 10−4) was significantly lower and the Km value (4.1 ± 0.5 mM) was significantly greater than that for CoMSH (Figure S2C). The results indicate that CoMSH is the preferred physiological reductant for MRX from M. acetivorans, in analogy to GSH as the reductant for GRXs in the domain Bacteria, although an additional role for CoASH cannot be ruled out. No activity was detected when MRX was assayed for HED-dependent deglutathionylation or reduction of dehydroascorbate. The midpoint potential for disulfide bond formation of MRX was determined by measuring fluorescence emission from the covalent adduct formed by the reaction of MRX with mBBr.22,31,32 The titration results (Figure 2) were fit to an n

Figure 3. Overall structure of MRX. Cartoon model of the tetramer. Cadmium ions are represented as magenta spheres. Active-site residues Cys30 and Cys33 are represented as yellow spheres.

monomer is similar, with the standard deviation between Cα atoms being 0.3 Å. As in a classical thioredoxin-glutaredoxin fold, the secondary structure of each monomer is composed of three α-helices surrounding a four-stranded antiparallel β-sheet. The α-helices and β-strands are flanked by eight loops. Two dimers of MRX form the tetramer and are held together through electrostatic interactions with eight cadmium ions at the N-termini (Figure 3). The two cadmium ions in each monomer are 4 Å apart and coordinate with the side chains of residues Asp7, Asp9, Asp7* (from an adjacent monomer), Lys12, Ser15, Asp18, and Asp46, bringing order to the Nterminal region. The tetramer assembly seems to be a feature of crystal packing, as dynamic light scattering indicated that it is a monomer in solution. Seventeen N-terminus residues (6−22) are predominantly hydrophilic in nature and form an extended loop region that faces the water- and cadmium-occupied core of the tetramer. These residues have several Ramachandran backbone ϕ,ψ outliers because of the backbone geometry distortion caused by the cadmium ions. The average B-factor of the N-terminal residues in particular and the protein tetramer

Figure 2. Redox titration of disulfide bond formation in MRX. The amplitude is on a scale where the amplitude measured at the most negative reduced value is a value of 100. Em (mV) represents actual potentials. Value reported in the text is the mean ± the standard deviation of biological triplicates.

= 2 Nernst equation with a midpoint potential (Em) of −201 ± 3 mV at pH 7.0. The value lies within the range of redox potentials (−198 to −233 mV at pH 7.0) reported for classical dithiol GRXs.33−35 CoMS-SCoM Reductase Activity. The finding that MRX utilizes CoMSH as an electron donor implies that CoMSSCoM is a product requiring reduction back to CoMSH. A NAD(P)H-dependent CoMS-SCoM reductase from the D

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successful in displacing the tightly bound cadmium ion, as no evidence for the CoMSH molecule could be seen in the electron density map. These results suggest the presence of cadmium precludes binding of CoMSH. Indeed, 1 mM cadmium chloride abolished enzyme activity. Structural Comparison of MRX with GRX. While MRX is 101 amino acids long, most GRXs are about 85 amino acids. Sequence alignment (Figure 5) shows that the extra length in MRX is due to an additional 22 residues in the N-terminal region. These residues have high disorder and are far from the active site. However, the overall fold of MRX and GRX1 from E. coli (PDB code 1GRX) is similar, supported by superposition with an RMS of 3.7 Å for 68 Cα atoms. The electrostatic potential in the substrate binding pocket of MRX (Figure 4A) is largely positive, similar to that of GRX1. The CVWC region of MRX superposes well with the CPYC region of GRX1 (Figure 4C), consistent with GSH as a reductant for MRX. Active-Site Modeling. In lieu of crystal structures with substrates bound, models of GSH and CoMSH (Figure 6) were built into the active site of MRX and energy minimized using the YASARA server.28 The recently solved crystal structures of cGRX2 from Clostridium oremlandii bound with GSH (PDB code 4TR1) and GSSG (PDB code 4TR0) were used for modeling.37 Although the overall and active-site architectures of MRX resemble that of GRX, substantial differences in binding of GSH were found.37−40 GRX structures share conserved GSH binding-site motifs consisting of CXXC, TVP, and C/NDD, whereas the corresponding motifs in MRX are CVWC (30− 33), SFP (75−77), and FKE (90−92) (Figure 5). The interactions that GSH has with the TVP motif are strictly conserved in all GRX structures, where the amide group of Val forms a bidentate hydrogen bond with the amide group close to the middle of GSH.37 GSH modeled in MRX (Figure 6A) forms a hydrogen bond with the amino nitrogen and carbonyl oxygen atoms of Phe76 in the SFP motif corresponding to Val in the TVP motif of GRXs (Figure 5). The modeled GSHbound structure also shows hydrogen bonding to the amino nitrogen atom of Phe90 in the FKE motif (Figure 6A), corresponding to Cys in the CDD motif of GRXs (Figure 5). The sulfur atom of GSH in MRX resides 2.9 Å from the sulfhydryl of Cys30 of the active-site C30VWC33 disulfide. CoMSH modeled in the active site of MRX shows the sulfonate moiety hydrogen bonded to the backbone amide and carbonyl oxygen of Phe76 (Figure 6B). At a distance of 2.8 Å,

in general is high, reflecting high disorder in the crystals (Table S1), which led to a moderate diffraction resolution of 2.45 Å. N-terminal residues 1−5 are disordered and not traceable in the electron density maps, so they are missing in the deposited PDB model. The surface rendition of MRX (Figure 4) shows a pocket containing a C30VWC33 motif, which identifies an active-site

Figure 4. Surface representation of MRX showing the active-site pocket. (A) Location of the CVWC motif (stick representation) and associated cadmium ion (yellow ball) in the active-site pocket. (B) Stereoview of electron density at the CVWC motif contoured at 1σ for cysteines and the cadmium ion. (C) Superposition of the active-site sequence CVWC in MRX (blue) and CPYS in E. coli GRX1 (orange).

architecture that is similar to that of GRXs and appropriate for binding substrates. A cadmium ion from the crystallization solution is seen coordinating with the reduced sulfhydryls of the C30VWC33 motif. Soaking crystals with CoMSH was not

Figure 5. Alignment of amino acid sequences of methanoredoxin (MRX) from Methanosarcina acetivorans with GSH-bound GRX structures. Escherichia coli GRX1 (AAA23936); Escherichia coli GRX3 (AIF96177); Saccharomyces cerevisiae GRX1 (CAA42381); Methanosarcina acetivorans MRX (NP_616586.1); and Clostridium ormelandii cGRX2 (4TR1_B). Residues implicated in binding GSH are in bold and italic. Hash marks indicate conserved motifs. Sequences were recovered from the NCBI database and aligned with COBALT. E

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homologues in clade III are consistently longer (90−103). Sequence identities in clade III ranged from 45 to 75%. The results suggest that MRX homologues are widespread in diverse methanogens. Additional sequences with lower identity to MRX were retrieved that lacked the dithiol GRX distinctive prolinecontaining active-site consensus sequence C[P/S][Y/F]C and more closely resembled the MRX active-site consensus (Figure S4). The sequences were from diverse nonmethanogenic prokaryotic species with sequence identities to MRX ranging from 31 to 59%. The results suggest that MRX-like proteins are present in nonmethanogen species.

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DISCUSSION Comparison of MRX and GRX. Although the overall fold and active-site architecture of MRX resembles that of GRXs, several other properties of MRX are distinct from GRXs. While MRX is 101 amino acids long, most GRXs are about 85 amino acids. Sequence identities are below 20%, and the consensus sequence of the active-site CXXC motif in MRX is C[V/I/G/ Q][W/H]C compared with the dithiol GRX consensus of C[P/S][Y/F]C. Proline is responsible for inducing structural features in proteins essential for efficient substrate binding and catalysis. Thus, the presence or absence of proline may play an important role in interactions with reductant and partner proteins specific for MRX and GRX. Finally, although common for GRXs, no HED-dependent deglutathionylation or dehydroascorbate reduction activities were observed for MRX.5 The results show that MRX and MRX homologues in clade III (Figure 7) are distinct from GRXs and unique to methanogens. The results also suggest that MRXs, although significantly different from GRXs, are expected to share an ancestor with GRXs from the domain Bacteria. This proposal is consistent with the finding that sequences from methanogens in clades I and II more closely resemble GRXs. Finally, the results call for the characterization of proteins in clades I and II to further understand the significance of GRX-like enzymes in methanogens. Physiological Significance. A role has been proposed for CoMSH in methanogens analogous to roles for GSH.17 Consistent with this proposal, CoMSH was a reductant in the insulin disulfide reductase assay of MRX. Furthermore, kinetic analyses of MRX revealed a Km for CoMSH (2.7 mM) similar to cytoplasmic levels of CoMSH reported for a diversity methanogens that averaged 2 mM.42 It was recently shown that levels of CoMSH increase 7-fold in response to cadmiuminduced oxidative stress, a result consistent with the proposed role for CoMSH.43 The proposed role is further supported by the finding that M. acetivorans cell-free extract reduces the product of MRX (CoMS-SCoM), regenerating CoMSH. CoASH also served as a reductant for insulin disulfide reductase activity of MRX, albeit less effectively than CoMSH. Although the levels of CoASH in methanogens have not been reported, it is doubtful that they equal the levels of CoMSH when considering CoASH functions in biosynthetic pathways as opposed to the more demanding energy conserving pathways of methanogens with the exception of acetotrophic species. Nonetheless, until in vivo concentrations of CoASH and CoASH are known, a thiol protective role for CoASH in addition to CoMSH cannot be ruled at this juncture. GRX-like enzymes utilizing a diversity of thiol protective systems other than GSH are common among prokaryotes of the domain Bacteria.16 Mycobacterium species produce milli-

Figure 6. Stick representation of active-site motifs showing hydrogenbond interactions with CoMSH and GSH as modeled in the active site of MRX. (A) Energy minimized model of GSH, as deduced from the GSH-bound structure of cGRX2 from C. oremlandii. (B) CoMSH. Loops containing Phe76 and Phe90 interact with GSH and CoMSH, forming three and two hydrogen bonds, respectively (black dotted lines). The dotted lines represent distances less than 3.4 Å.

the sulfhydryl of CoMSH is oriented toward the sulfhydryl of Cys30 of the active-site C30VWC33 disulfide similar to GSH. The results suggest a catalytic mechanism similar to that of GRXs in which either reductant attacks Cys30, forming the mixed disulfide and the sulfhydryl of Cys33.41 The mixed disulfide is attacked by the second reductant molecule, producing CoMSSCoM or GSSG and the sulfhydryl of Cys33, thereby readying the enzyme for disulfide exchange with the protein substrate. Bioinformatics Analyses. The gene (MA1658) encoding MRX from M. acetivorans is annotated as a GRX, although the sequence identity with GRXs from the domains Bacteria and Eukarya with a GRX consensus C[P/S][Y/F]C active-site motif is only 18 and 17%, respectively (Figure 5). A BLASTp search was conducted with M. acetivorans MRX as the query while constraining searches to proteins with the CXXC motif and lengths of 77−116 amino acids. The results revealed MRX homologues in 34 of 67 methanogen genomes represented in the NCBI nonredundant database (Table S2). Approximately half of the species contained two MRX homologs, and the other half, contained one. Metabolically diverse methanogens were represented, as were all phylogenetic orders except Methanopyrales, results similar to those reported for TRXs of methanogens.13 A distance tree of significant alignments revealed three major clades of MRX homologues for which clade III was closer to MRX than clades I and II, which were closer to GRX1 from E. coli (Figure 7). A multiple alignment of clade III sequences (Figure S3) showed a consensus sequence of C[V/I/G/Q][W/H]C distinct from the dithiol GRX consensus of C[P/S][Y/F]C.1 However, clades I and II (Figure S3) showed consensus sequences of CP[N/K]C and CP[W/ F]C, resembling the dithiol GRX consensus from the domains Bacteria and Eukarya. Furthermore, the lengths of sequences in clades I and II (82−90 residues) resemble GRXs, whereas MRX F

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Biochemistry

Figure 7. Distance tree of glutaredoxin annotations from methanogens and GRX1 from E. coli. The tree was constructed using the neighbor-joining method with significant alignments recovered from the NCBI database queried with MRX from M. acetivorans. The scale bar indicates the average number of amino acid substitutions per site. Key: Methanosarcina acetivorans C2A, NP_616586.1; Methanoplanus petrolearius DSM11571 (1), WP_013329188.1; Methanosarcina mazei Gö1 (1), YP_007490064.1; Methanosarcina barkeri Fusaro (1), YP_304586.1; Methanococcoides methylutens, KGK99245.1; Methanococcoides burtonii, WP_011500136.1; Methanocella paludicola (1), WP_012899727.1; Methanocella arvoryzae MRE50 (1), YP_687352.1; Methanohalobium evestigatum Z-7303, YP_003727821.1; Methanosalsum zhilinae DSM4017, YP_004617015.1; Methanolobus psychrophilus R15, YP_006922927.1; Methanosarcina mazei Gö1 (2), YP_007491892.1; Methanosarcina barkeri Fusaro (2), YP_305929.1; Methanosaeta harundinacea 6Ac, YP_005921093.1; Methanosaeta concilii GP6, YP_004382764.1; Methanobacterium lacus, WP_013646029.1; Methanobacterium sp. MBC34, WP_008512547.1; Methanobacterium formicicum, AIS32944.1; Methanobacterium sp. SWAN-1, WP_013824719.1; Methanothermobacter thermautotrophicus, WP_010877185.1; Methanothermobacter marburgensis Marburg, YP_003849085.1; Methanoculleus marisnigri JR1, YP_001048358.1; Methanoculleus bourgensis MS2, YP_006546214.1; Methanoregula formicica, YP_007249622.1; Methanoregula boonei 6AB, YP_001403993.1; Methanoplanus limicola DSM2279, EHQ34301.1; Methanosphaerula palustris E1-9c, WP_012617193.1; Methanofollis liminatans DSM4140, EJG07388.1; Methanospirillum hungatei JF-1, YP_502397.1; Methanocella paludicola (2), WP_012901144.1; Methanocella arvoryzae MRE50 (2), YP_686873.1; Methanoplanus petrolearius DSM2279 (2), YP_003894539.1; Methanoculleus sp. MH98A, KDE55450.1; Escherichia coli K12, AAA23936.1.

oxidative stress similar to GSH and GRXs of the domain Bacteria.4 Although GSH was a reductant for insulin disulfide reduction by MRX, a role for GSH in M. acetivorans and all other methanogens is questionable when considering that a database search of the 67 methanogen genomes retrieved only two species with annotations for the glutathione (GSSG) synthesis enzymes glutamate-cysteine ligase and glutathione synthase that did not include M. acetivorans (Table S3). Furthermore, an alternative role is reported for the ligase and synthase homologues in the synthesis of methanogen cofactors.48 Although it has been reported that glutathione reductase activity is absent in Methanosarcina barkeri, annotations were

molar concentrations of mycothiol as reductant for the GRXlike enzyme mycoredoxin-1 that contains the CGYC active site and participates in oxidative stress response.44 Microredoxin also functions in Corynebacterium glutamicum to shuttle electrons between mycothiol and arsenate reductase in analogy to a role for GSH and GRXs of E. coli.45 Bacillithiol is a glutathione surrogate of Bacillus subtilis and other firmicutes.46,47 Thus, it is not surprising that CoMSH functions as a thiol protectant in M. acetivorans and doubtless in other phylogenetically and metabolically diverse methanogens where CoMSH is universal. It follows that CoMSH and MRX are expected to play important roles in redox control, signaling, and G

DOI: 10.1021/acs.biochem.5b00823 Biochemistry XXXX, XXX, XXX−XXX

Biochemistry

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ABBREVIATIONS MRX, methanoredoxin; CoMSH, coenzyme M (2-mercaptoethanesulfonate); CoBSH, coenzyme B; CoASH, coenzyme A; GSSG, oxidized glutathione; GSH, reduced glutathione; HED, β-hydroxyethyl disulfide; RMSD, root-mean-square deviation; mBBr, monobromobimane

retrieved from the databases for putative glutathione reductases in seven methanogens, including Methanosarcina species.30 Concluding Remarks. Although it is well-documented that TRXs and GRXs are widespread and of fundamental importance in organisms from the domains Bacteria and Eukarya, little is understood of these proteins from the domain Archaea, which includes methanogens. Previous investigations from Archaea have been limited to TRXs. The results reported here describe a GRX-like enzyme (methanoredoxin, MRX), abundant in metabolically and phylogenetically diverse methanogens, with properties distinct from those of GRXs of the domains Bacteria and Eukarya. Importantly, the work identifies a novel role for the universal methanogen cofactor CoMSH that likely also plays roles in the redox physiology of methanogens.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b00823. SDS-PAGE gel of MRX (Figure S1), double-reciprocal plots supporting kinetic data (Figure S2), sequence alignments of MRX homologues (Figures S3 and S4), crystallographic data-collection statistics (Table S1), putative MRXs in methanogens (Table S2), and enzymes related to glutathione synthesis and reduction in methanogens (Table S3) (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*(N.H.Y.) Tel.: (814) 863-9387. E-mail: [email protected]. *(H.P.Y.) Tel.: (814) 865-8383. E-mail: [email protected]. *(J.G.F.) Tel.: (814) 863-5721. E-mail: [email protected]. Present Address §

(A.K.K.) Sardar Patel Renewable Energy Research Institute, Vallabh Vidyanagar, Anand 388120, Gujarat, India. Author Contributions ∥

D.P. and D.Y. contributed equally to this work. D.P., D.Y., A.K.K., R.S.S.K., and J.G.F. designed the research; D.Y., D.P., and R.S.S.K. performed biochemical experiments; D.Y., N.H.Y., and H.P.Y. designed crystallographic experiments and solved the crystal structure; D.Y., D.P., N.H.Y., H.P.Y., R.S.S.K., and J.G.F. wrote the paper; all authors helped with the final draft. Funding

This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through grant DE-FG0295ER20198 MOD16 to J.G.F. National Institutes of Health− National Center for Research Resources shared instrumentation grant 1S10RR023439-01 was awarded to N.H.Y. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Tatiana Laremore for the LC-MS analysis of MRX. We very much appreciated the helpful discussions and technical assistance given by Eric Malmberg and Venkata Vepachedu. H

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DOI: 10.1021/acs.biochem.5b00823 Biochemistry XXXX, XXX, XXX−XXX