Methyl-coenzyme M reductase (MCR) is a unique enzyme found exclusively in anaerobic archaea, where it catalyzes the reversible conversion of methyl-coenzyme M (CoM, 2-methylmercaptoethanesulfonate) and coenzyme B (CoB, 7-thioheptanoylthreoninephosphate) to methane and a CoB-CoM heterodisulfide (Ermler et al., 1997; Scheller et al., 2010):

This reaction, which has been proposed to proceed via an unprecedented methyl radical intermediate (Wongnate et al., 2016), plays a critical role in the global carbon cycle (Thauer et al., 2008). In the forward direction, MCR catalyzes the formation of methane in methane-producing archaea (methanogens). In the reverse direction, MCR catalyzes the consumption of methane in methanotrophic archaea (known as ANMEs, for anaerobic oxidation of methane). Together, these processes produce and consume gigatons of methane each year, helping to establish the steady-state atmospheric levels of an important greenhouse gas. MCR displays an α₂β₂γ₂ protein domain architecture and contains two molecules of F₄₃₀, a nickel porphinoid cofactor (Ermler et al., 1997; Zheng et al., 2016; Moore et al., 2017). The reduced Ni(I) form of F₄₃₀ is essential for catalysis (Goubeaud et al., 1997), but is highly sensitive to oxidative inactivation, a feature that renders biochemical characterization of MCR especially challenging. As a result, many attributes of this important enzyme remain uncharacterized.

An unusual feature of MCR is the presence of several modified amino acids within the active site of the α-subunit. Among these are a group of methylated amino acids, including 3-methylhistidine (or N¹-methylhistidine), S-methylcysteine, 5(S)-methylarginine, and 2(S)-methylglutamine, which are presumed to be installed post-translationally by S-adenosylmethionine-dependent methyltransferases (Selmer et al., 2000; Kahnt et al., 2007). 3-methylhistidine is found in all MCRs examined to date, whereas the presence of the other methylated amino acids is variable among methane-metabolizing archaea (Kahnt et al., 2007). A didehydroaspartate modification is also observed in some, but not all, methanogens (Wagner et al., 2016). Lastly, a highly unusual thioglycine modification, in which the peptide amide bond is converted to a thioamide, is present in all methanogens that have been analyzed to date (Figure 1—figure supplement 1) (Ermler et al., 1997; Kahnt et al., 2007).

The function(s) of the modified amino acids in MCR has/have not yet been experimentally addressed; however, several theories have been postulated. The 3-methylhistidine may serve to position the imidazole ring that coordinates CoB. This methylation also alters the pK_a of histidine in a manner that would promote tighter CoB-binding (Grabarse et al., 2000). The variable occurrence of the other methylated amino acids suggests that they are unlikely to be directly involved in catalysis. Rather, it has been hypothesized that they tune enzyme activity by altering the hydrophobicity and flexibility of the active site pocket (Kahnt et al., 2007; Grabarse et al., 2000). A similar argument has been made for the didehydroaspartate residue (Wagner et al., 2016). In contrast, three distinct mechanistic hypotheses have implicated the thioglycine residue in catalysis. One proposal is that the thioglycine facilitates deprotonation of CoB by reducing the pK_a of the sulfhydryl group (Grabarse et al., 2001). A second proposal suggests that thioglycine could serve as an intermediate electron carrier for oxidation of a proposed heterodisulfide anion radical intermediate (Horng et al., 2001). Lastly, cis-trans isomerization of the thioamide during catalysis could potentially play an important role in coupling the two active sites via a previously proposed two-stroke mechanism for the enzyme (Goenrich et al., 2005).

Thioamides are rare in biology. While cycasthioamide is plant-derived (Pan et al., 1997), most other naturally occurring thioamides are of bacterial origin. Among these are the ribosomally synthesized and post-translationally modified (RiPP) peptide natural products thioviridamide (Hayakawa et al., 2006) and methanobactin (Kim et al., 2004; Kenney and Rosenzweig, 2012; Kenney and Rosenzweig, 2013), as well as closthioamide, which appears to be an unusual non-ribosomal peptide (Lincke et al., 2010; Chiriac et al., 2015). The nucleotide derivatives thiouridine and thioguanine (Coyne et al., 2014), and two additional natural product antibiotics, thiopeptin and Sch 18640 (Puar et al., 1981; Hensens and Albers-Schönberg, 1983), also contain thioamide moieties. Although the mechanism of thioamide installation in peptides has yet to be established, the identification of the thioviridamide biosynthetic gene cluster provides clues to their origin (Izawa et al., 2013). Two of the proteins encoded by this gene cluster have plausible roles in thioamide synthesis. The first, TvaH, is a member of the YcaO superfamily, while the second, TvaI, is annotated as a ‘TfuA-like’ protein (Izawa et al., 2013). Biochemical analyses of YcaO-family proteins indicate that they catalyze the ATP-dependent cyclodehydration of Cys, Ser, and Thr residues to the corresponding thiazoline, oxazoline, and methyloxazoline (Figure 1). Many ‘azoline’ heterocycles undergo dehydrogenation to the corresponding azole, which are prominent moieties in various RiPP natural products classes, such as linear azol(in)e-containing peptides, thiopeptides, and cyanobactins (Dunbar et al., 2012; Dunbar et al., 2014; Arnison et al., 2013; Burkhart et al., 2017). The YcaO-dependent synthesis of peptidic azol(in)e heterocycles often requires a partner protein, which typically is the neighboring gene in the biosynthetic gene cluster (Burkhart et al., 2015; Dunbar et al., 2015; Wright et al., 2006). Based on enzymatic similarity, the TfuA protein encoded adjacent to the YcaO in the thioviridamide pathway may also enhance the thioamidation reaction, perhaps by recruiting a sulfurtransferase protein. Although not RiPPs, thiouridine and thioguanine biosynthesis requires the use of sulfurtransferases (Coyne et al., 2013; Palenchar et al., 2000).

Figure 1 with 1 supplement see all

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The biosynthesis of the thioglycine in MCR was proposed to occur by a mechanism similar to that used to produce thioamide-containing natural products (Kahnt et al., 2007), a prediction made six years prior to the discovery of the thioviridamide biosynthetic gene cluster (Izawa et al., 2013). Given their putative role in thioamidation reactions, it is notable that YcaO homologs were found to be universally present in an early analysis of methanogen genomes, resulting in their designation as ‘methanogenesis marker protein 1’ (Basu et al., 2011). Genes encoding TfuA homologs are also ubiquitous in methanogens, usually encoded in the same locus as ycaO, similar to their arrangement in the thioviridamide biosynthetic gene cluster. Therefore, both biochemical and bioinformatic evidence are consistent with these genes being involved in thioglycine formation. In this report, we use the genetically tractable methanogen Methanosarcina acetivorans to show that installation of the thioamide bond at Gly₄₆₅ in the α-subunit of MCR requires the ycaO-tfuA locus (Figure 1—figure supplement 1). As MCR is essential for growth and survival (Rother et al., 2005), the viability of ycaO-tfuA mutants precludes the hypothesis that the thioamide residue is essential for MCR activity. Instead, our phenotypic analyses support a role for thioglycine in maintaining a proper structural conformation of residues near the active site, such that absence of this modification in MCR might be particularly detrimental on growth substrates that have low free energy yields as well as under additionally destabilizing conditions such as elevated temperatures.

The loss of the thioglycine modification in the ∆ycaO, ∆tfuA, and ΔycaO-tfuA mutants shows that both of these genes are required for the thioamidation of McrA. Although this could be an indirect requirement, we believe it is more likely that the YcaO/TfuA proteins directly catalyze modification of McrA. This conclusion is mechanistically compatible with biochemical analyses of YcaO homologs. YcaO enzymes that carry out the ATP-dependent cyclodehydration of beta-nucleophile-containing amino acids have been extensively investigated (Burkhart et al., 2017). Such cyclodehydratases coordinate the nucleophilic attack of the Cys, Ser, and Thr side chain on the preceding amide carbonyl carbon in a fashion reminiscent of intein splicing (Perler et al., 1997) (Figure 1). The enzyme then O-phosphorylates the resulting oxyanion and subsequently N-deprotonates the hemiorthoamide, yielding an azoline heterocycle. An analogous reaction can be drawn for the YcaO-dependent formation of peptidic thioamides. The only difference is that an exogenous equivalent of sulfide is required for the thioamidation reaction, rather than an adjacent beta-nucleophile-containing amino acid for azoline formation (Figure 1).

Most YcaO cyclodehydratases require a partner protein for catalytic activity. The earliest investigated YcaO partner proteins are homologs of the ThiF/MoeB family, which are related to E1 ubiquitin-activating enzymes (Dunbar et al., 2014; Schulman et al., 2009). These YcaO partner proteins, as well as the more recently characterized ‘ocin-ThiF’ variety (Dunbar et al., 2015), contain a ~ 90 residue domain referred to as the RiPP precursor peptide Recognition Element (RRE), which facilitates substrate recognition by interacting with the leader peptide. Considering these traits of azoline-forming YcaOs, it is possible that thioamide-forming YcaOs require the TfuA partner to facilitate binding to the peptidic substrate. Alternatively, TfuA may recruit and deliver sulfide equivalents by a direct or indirect mechanism. In this regard, it is noteworthy that the ycaO-tfuA locus can be found adjacent to genes involved in sulfur and molybdoterin metabolism (Figure 2). Many of these genes encode proteins with rhodanese-like homology domains, which are well-established sulfurtransferases. These enzymes typically carry sulfur in the form of a cysteine persulfide, a non-toxic but reactive equivalent of H₂S (Palenchar et al., 2000; Matthies et al., 2005). Akin to rare cases of azoline-forming, partner-independent YcaOs, certain methanogens lack a bioinformatically identifiable TfuA (e.g. Methanopyrus kandleri and Methanocaldococcus sp., Figure 2). Whether these YcaOs act independently or use an as yet-unidentified partner protein remains to be seen. Clearly, further in vitro experimentation will be required to delineate the precise role of TfuA in the thioamidation reaction.

The viability of the ΔycaO-tfuA mutant raises significant questions as to the role of thioglycine in the native MCR enzyme, especially considering its universal presence in all MCRs examined to date. We considered three hypotheses to explain this result. First, we examined the possibility that thioglycine modification is involved in enhancing the reaction rate (Kahnt et al., 2007; Horng et al., 2001). Although it has not been explicitly determined, MCR is thought to catalyze the rate-limiting step of methanogenesis (Scheller et al., 2010; Wongnate et al., 2016). Therefore, the absence of thioglycine might lead to a corresponding decrease in the growth rate, with more pronounced defects on substrates that lead to the fastest growth. However, we observed the opposite: the most pronounced defects were observed with growth substrates that support the slowest WT growth rates (Table 1). Next, we considered the possibility that the thioglycine influences substrate affinity. C₁ units enter methanogenesis at the level of N⁵-methyl-tetrahydrosarcinapterin (CH₃-H₄SPt) during growth on acetate (Galagan et al., 2002; Deppenmeier et al., 1999), but at the level of methyl-CoM during growth on DMS, methanol, and TMA (Figure 6) (Bose et al., 2008; Fu and Metcalf, 2015). Significantly, these entry points are separated by an energy-dependent step that is coupled to production or consumption of the trans-membrane Na⁺ gradient. As a result, intracellular levels of methyl-CoM, CoM, and CoB could possibly be significantly different depending on the entry point into the methanogenic pathway. Because we observed growth defects on substrates that enter at both points (i.e. DMS and acetate), we suspect that the growth deficiency phenotype is unlikely to be related to changes in substrate affinity. A third explanation we considered was that thioglycine increases the stability of MCR. In this model, unstable MCR protein would need to be replaced more often, creating a significant metabolic burden for the mutant. Consistent with our results, this additional burden would be exacerbated on lower energy substrates like DMS and acetate, especially given that MCR comprises ~10% of the total protein (Rospert et al., 1990). Further, one would expect that a protein stability phenotype would be exaggerated at higher temperatures, which we observed during growth on methanol (Figure 5). Thus, multiple lines of evidence support the idea that the growth-associated phenotypes stemming from the deletion of TfuA and YcaO are caused by decreased MCR stability. However, the melting temperature of MCR from the ∆ycao-tfuA mutant was modestly higher than that purified from the WT. As the thioglycine is buried deep within the active site of MCR (Ermler et al., 1997), these data are consistent with the notion that thioamidation does not affect the global stability of the protein. Instead, it suggests that the thioglycine modification impacts the local stability in the vicinity of the buried active site of MCR (Figure 1—figure supplement 1), which in turn might have a detrimental effect under catalytic turnover conditions.

Figure 6

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The chemical properties of amides relative to thioamides are consistent with our hypothesis. Although amides tend to be planar, their rotational barriers are lower than for the corresponding thioamide and thus they are more conformationally flexible. This is especially true for glycine. Considering the negligible electronegativity difference between sulfur and carbon, the thioamide carbonyl is not polarized like an amide carbonyl (Wiberg and Rablen, 1995). Further, sulfur has a larger van der Waals radius than oxygen resulting in a thioamide bond length that is ~40% longer than the amide bond (1.71 Å versus 1.23 Å) (Petersson et al., 2014), which presents additional steric hindrances to backbone flexibility. Finally, the pK_a of thioamides is lower than amides, making thioglycine a stronger hydrogen bond donor than glycine (Lee et al., 2002), which again could reduce conformational flexibility. Taken together, it is chemically reasonable to state that the increased flexibility of the unmodified glycine in the ΔycaO-tfuA mutant would render the contorted conformation of the peptide backbone in the Gly₄₆₂-Leu₄₆₉ region of McrA considerably less stable (Figure 1—figure supplement 1). Indeed, this region adopts a rather strange configuration, with the side chain of Phe₄₆₃ bending back towards the thioamide of Gly₄₆₅. The molecular alignment and distance (3.5 Å) suggests a potential π-cation interaction between these residues(Figure 1—figure supplement 1) The thioamide sulfur is also optimally distanced (3.6 Å) and geometrically positioned to form an H-bond with the side chain of Asn₅₀₁ and the backbone N-H of Leu₄₆₉. There also appears to be a hydrophobic interaction between the thioamide and the beta carbon of Leu₄₆₉, which would be energetically unfavorable with a more polarized amide. These observations suggest that the local stability in the vicinity of the active site imparted by the thioamide is required for protein stability and increased half-life of the MCR protein. A conclusive test of this hypothesis will require comprehensive structural and biochemical characterization of the active MCR variant lacking the thioglycine modification, which is beyond the scope of this work.

Finally, the temperature-sensitive phenotype of the ΔycaO-tfuA mutant has potential implications regarding the evolution and ecology of methanogenic archaea. Based on this result, it seems reasonable to speculate that the thioglycine modification would be indispensable for thermophilic methanogens. It is often posited that the ancestor of modern methanogens was a thermophilic organism (Gribaldo and Brochier-Armanet, 2006; Forterre, 2015; Weiss et al., 2016; López-García et al., 2015). If so, one would expect the thioglycine modification to be present in most methanogenic lineages, being stochastically lost due to genetic drift only in lineages that grow at low temperatures where the modification is not required. In contrast, if methanogenesis evolved in a cooler environment, one might expect the distribution of the modification to be restricted to thermophilic lineages. Thus, the universal presence of the thioglycine modification supports the thermophilic ancestry of methanogenesis. Indeed, the ycaO-tfuA locus is conserved even in Methanococcoides burtonii, a psychrophilic methanogen isolated from Ace Lake in Antarctica, where the ambient temperature is always below 2°C (Franzmann et al., 1992). It will be interesting to see whether this modification is maintained by other methanogenic and methanotrophic archaea growing in low temperature environments.

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[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea" for consideration by eLife. Your article has been reviewed by four peer reviewers and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Dianne K Newman (Reviewer #1) and Rudolph Thauer (Reviewer #4).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife at this time.

While there is sincere enthusiasm for your work, and a desire to see it ultimately published in eLife, the consensus opinion is that, as the manuscript currently stands, the key finding - genetic demonstration that a thioglycine in MCR is installed by YcaO/TfuA and is not essential for catalytic activity - is not a sufficient advance to appeal to the broad eLife audience. Though we appreciate that comparing the catalytic activity of WT and mutant MCR is well beyond the scope of the manuscript, some other biochemical follow-up would significantly strengthen other claims of the manuscript (e.g. thermostability, mechanism of YcaO/TfuA, role of TfuA, etc). The evolutionary implications are intriguing, but not yet convincing. For example, why would MCR from Methanopyrus kandleri growing above 100°C and psychrophilic methanogens and methanotrophs both have the thioglycine in the active site of MCR conserved? And why would the thioglycine only show up in MCR? If you are able and willing to perform some additional experiments to bolster the claims in the manuscript, we would be happy to reconsider it for eLife.

Reviewer #1:

This manuscript presents strong evidence that 2 genes (tfuA and ycaO) are required for installation of a thioglycine into McrA, a critical enzyme in methanogenesis and in organisms that oxidize methane anaerobically. These processes are globally significant and challenging to study, so work that improves our understanding of how they function is attractive for publication in eLife.

Positives of the paper include clear writing and well-done genetic and MS experiments. The application of biochemical understanding from the natural product field to identify putitive genes catalyzing installation of a thioglycine in a methanogen is elegant and chemically satisfying. However, while the authors provide physiological data and reasonable logic arguing that the thioglycine confers greater stability to MCR, the evidence supporting this claim is indirect. One concern is that there is some other target of the modification other than McrA that is responsible for the observed phenotype. Perhaps this can be excluded as a possibility, but if so, the authors need to explain why that is. To a naïve reader, this seems like an important missing piece. One option would be to perform a limited set of biochemical experiments to look at McrA stability with and without the thioglycine residue. The authors argue this is beyond the scope of the paper, but is it really? Could this be done by looking for different sized bands of McrA on a western blot (might one predict differential stability would show up in different patterns on a protein gel)? How difficult is it to purify McrA and test for temperature stability for the WT and mutant versions? Alternatively, could a different mutation be introduced that would be predicted to render the McrA protein less thermostable, and could the phenotype be recapitulated? In the absence of biochemical data, that might strengthen the argument. How can the authors be confident that other proteins produced under the experimental conditions are not modified by TfuA/YcaO and responsible for the phenotypes? At a minimum, the authors need to directly address this concern in their discussion.

The Abstract and Discussion end with speculation about the implications of this work for an evolutionary hot start for methanogenesis. This is a bold claim, and the phylogenetic evidence is intriguing, but for this claim to be convincing, it is important to have solid evidence that the thioglycine in McrA is what causes the lack of thermostability. I would like to see this manuscript published in eLife, but encourage the authors to perform another experiment to strengthen this critical point.

Reviewer #2:

This paper addresses the biosynthesis and functional significance of a post-translational modification found in methyl-coenzyme M reductase, an enzyme that produces methane in anaerobic archaea and may also be involved in methane oxidation by methanotrophic archaea. Several post-translationally modified residues are present in or near the MCR active site in the McrA subunit, including a thioglycine. Candidate proteins for producing this thioamide include a YcaO homolog paired with a TfuA homolog. YcaOs catalyze formation of thiazolines from cysteines, and TfuA-like proteins are hypothesized to play a role in sulfur delivery and/or substrate recognition. Bioinformatic analysis presented here supports a role for these proteins in McrA thioamidation, and genetic disruption of both genes in Methanosarcina acetivorans coupled with mass spectrometric analysis indicates that the thioglycine is not formed in their absence. While the cells are still viable, some differences in growth are observed between the wildtype and ΔycaO-tfuA strains. In particular, the knockout strain grows more slowly on DMS and acetate, and more slowly on methanol if the temperature is raised. From these growth data, the authors suggest that the thioglycine modification does not affect MCR catalysis or substrate affinity, but instead increases its thermal stability. Although this model is consistent with the results, biochemical characterization of the MCR is essential to addressing this hypothesis. In addition, forming a thioamide seems like a metabolically expensive way to increase thermostability as many other proteins are thermostable without such post-translational modifications. The authors note that biochemical work is beyond the scope of this study, but I am not sure that the knockout alone merits publication in eLife; it seems more suited to a specialized journal.

In terms of thinking about the modification and its potential role in thermal stability or other aspects of function, it would be helpful to have a picture of the MCR active site showing where the glycine residue is located and what interactions it has with other residues.

Reviewer #3:

Metcalf et al. address interesting questions regarding post-translational modifications of the last enzyme of the methanogenic pathway in archaea. It has been known for almost two decades that some residues in the active site of the methyl-coenzyme M reductase (Mcr) are modified. Of particular interest is the thioamidation of glycine. The goal was to identify genes encoding enzymes responsible for this modification. The authors build a case for the analysis of two genes (ycaO, tfuA) that tracked in some cases immediately upstream to the Mcr operon. The authors use Methanosarcina acetivorans to perform phenotypic analyses of strains with deletions of the genes of interest. The observed growth phenotypes are explained on the basis of the entry point of different substrates into the methanogenic pathway. The results are consistent with the explanations provided. However, the authors do not validate the presumed biochemical activities of YcaO or TfuA, hence leading to much speculation. The effects reported here could be indirectly leading to the absence of thioamidated glycine. The authors start the Discussion section by stating '[…] these genes (ycaO, tfuA) are directly involved in the post-translational modification of McrA'; I disagree. To support such a statement the authors must show that YcaO/TfuA can modify McrA in vitro.

Reviewer #4:

The manuscript by Metcalf et al. for eLife describes the important finding that the post-translational installation of thioglycine in methyl-coenzyme M reductase in the methanogenic archaeon Methanosarcina acetivorans is mediated by the tfuA-ycaO locus. This experimental procedure showing this is perfect and the results obtained convincing. The excellent manuscript should be published after considering the following minor points:

Discussion paragraph three and following: Yes, MCR is generally thought to be the rate limiting enzyme in methanogenesis, however, the only step in methanogenesis that is physiologically irreversible, is the heterodisulfide reductase reaction rather than the MCR reaction. Thus MCR is most probably not rate limiting. This should be considered or at least discussed.

Of the many glycins in McrA only one is converted to a thioglycin and this glycine is buried deep within the McrABC complex. The post-translational modification must therefore occur as long as the protein is not yet folded. Generally enzymes mediating post-translational modifications specifically recognize the sequence upstream and downstream of the amino acid to be modified. Could the authors comment on this in the manuscript with a few sentences?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea" for further consideration at eLife. Your revised article has been favorably evaluated by Gisela Storz (Senior editor) and four reviewers, one of whom is a member of our Board of Reviewing Editors.

We appreciate your efforts to respond to our feedback by performing new experiments and revising the text accordingly. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The consensus is that in the absence of supporting biochemical data, the manuscript still pushes a mechanistic message too strongly. Please moderate the discussion. Your results have shown that the thioglycine within the active site is not essential for full activity, not that it lacks a direct catalytic role. In addition, the paragraph on the discussion of growth phenotypes is convoluted and overly speculative. It would be better to tighten and moderate this paragraph, in the absence of supporting data. Growth phenotypes should also be mentioned in the Abstract, so it is clear that the phenotype of the double mutant extends beyond the lack of a PTM. While we agree that additional biochemical experiments are outside the scope of this work, for this manuscript to be published in eLife as a genetics/physiology paper, these aspects need to be airtight. Towards that end, please include complementation experiments for the mutants.

With these modifications, we anticipate your second revision would be publishable in eLife.

Reviewer #1:

The new experiments and revisions to the text have alleviated my previous concerns and the manuscript is much improved.

While there are many questions remaining that need to be addressed at the biochemical level, this study has made an important new contribution and is done rigorously.

Reviewer #2:

This is a revised version in which the authors have addresses most of the questions and suggestions made by the reviewers. The interpretations remain speculative but the experimental results are too important not to be published.

Reviewer #3:

In the revised version of this manuscript, Metcalf et al. show that strains carrying null alleles of tfuA or ycaO do not modify the glycine of interest. In addition, results from new experiments show that thioglycine is not needed to stabilize MCR. The new hypothesis is now that the stabilizing effect occurs at the level of the active site; that idea was not tested. In the absence of experimental data, the authors softened the wording regarding the direct involvement of TfuA and YcaO.

Without a question, this is a very interesting problem, but I am not convinced that the revised paper has reached the level of an eLife publication.

Reviewer #4:

In this revised manuscript, the authors have added two experiments. First, they have constructed separate knockouts of ycaO and tfuA as well as the double knockout, and demonstrate by mass spectrometric analysis that both are necessary for thioglycine formation. This is not surprising. Second, they have isolated the MCR produced by the double knockout strain and show that its melting temperature is unchanged, disproving their previous hypothesis regarding thermal stability. They have revised the hypothesis to invoke localized stability at the active site; the overall argument regarding the properties of amides versus thioamides is the same. A stability experiment is one of the approaches the editor suggested as biochemical follow up so in that sense, the authors have done what was requested.

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

This manuscript presents strong evidence that 2 genes (tfuA and ycaO) are required for installation of a thioglycine into McrA, a critical enzyme in methanogenesis and in organisms that oxidize methane anaerobically. These processes are globally significant and challenging to study, so work that improves our understanding of how they function is attractive for publication in eLife.

Positives of the paper include clear writing and well-done genetic and MS experiments. The application of biochemical understanding from the natural product field to identify putitive genes catalyzing installation of a thioglycine in a methanogen is elegant and chemically satisfying. However, while the authors provide physiological data and reasonable logic arguing that the thioglycine confers greater stability to MCR, the evidence supporting this claim is indirect. One concern is that there is some other target of the modification other than McrA that is responsible for the observed phenotype. Perhaps this can be excluded as a possibility, but if so, the authors need to explain why that is. To a naïve reader, this seems like an important missing piece. One option would be to perform a limited set of biochemical experiments to look at McrA stability with and without the thioglycine residue. The authors argue this is beyond the scope of the paper, but is it really? Could this be done by looking for different sized bands of McrA on a western blot (might one predict differential stability would show up in different patterns on a protein gel)? How difficult is it to purify McrA and test for temperature stability for the WT and mutant versions? Alternatively, could a different mutation be introduced that would be predicted to render the McrA protein less thermostable, and could the phenotype be recapitulated? In the absence of biochemical data, that might strengthen the argument. How can the authors be confident that other proteins produced under the experimental conditions are not modified by TfuA/YcaO and responsible for the phenotypes? At a minimum, the authors need to directly address this concern in their discussion.

We thank the reviewer for their insightful critique. We recognize that we had not rigorously shown that the in vivo phenotype resulting from the ycaO-tfuA deletion is due to an affect on MCR (rather than on some unidentified target of ycaO/tfuA) and we have made a dedicated effort to address this. To do this, we introduced affinity tag at the N-terminus of the mcrG CDS in the WT strain as well as in a strain lacking the ycaO-tfuA locus in order to aid purification and studies of MCR. (As a side note, this is the first time affinity purification of MCR has been conducted in the native host.) A SYPRO-orange based Thermofluor assay was performed with MCR purified aerobically from each of these strains (Figure 5—figure supplement 1) to determine the melting temperature of the complex containing all three subunits. We did not observe any significant difference in the melting temperature of the MCR complex lacking thioglycine under these conditions. Also, the content of F430 cofactor was unchanged between WT and mutant as determined by UV-Vis spectroscopy.

The outcome of these critical experiments suggests that thioglycine does not affect the global stability of the MCR complex; however, local instability in the region of the MCR active site, which is buried within the α subunit (McrA), cannot be confirmed nor refuted at this time. While we are interested in determining the precise role of the thioglycine, the requisite anaerobic biochemical interrogation of the protein is beyond the scope of the current study.

In addition, a comprehensive proteomic analysis might determine if YcaO/TfuA modify other proteins, but it would be impossible to obtain 100% coverage of all peptides in such an experiment. Therefore, a negative result would still be inconclusive. Given the associated cost and effort required to conduct such an experiment, we feel this is not worth pursuing at this time.

The Abstract and Discussion end with speculation about the implications of this work for an evolutionary hot start for methanogenesis. This is a bold claim, and the phylogenetic evidence is intriguing, but for this claim to be convincing, it is important to have solid evidence that the thioglycine in McrA is what causes the lack of thermostability. I would like to see this manuscript published in eLife, but encourage the authors to perform another experiment to strengthen this critical point.

Given the inconclusive data cited above, we have significantly softened this argument in the revised version.

Reviewer #2:

This paper addresses the biosynthesis and functional significance of a post-translational modification found in methyl-coenzyme M reductase, an enzyme that produces methane in anaerobic archaea and may also be involved in methane oxidation by methanotrophic archaea. Several post-translationally modified residues are present in or near the MCR active site in the McrA subunit, including a thioglycine. Candidate proteins for producing this thioamide include a YcaO homolog paired with a TfuA homolog. YcaOs catalyze formation of thiazolines from cysteines, and TfuA-like proteins are hypothesized to play a role in sulfur delivery and/or substrate recognition. Bioinformatic analysis presented here supports a role for these proteins in McrA thioamidation, and genetic disruption of both genes in Methanosarcina acetivorans coupled with mass spectrometric analysis indicates that the thioglycine is not formed in their absence. While the cells are still viable, some differences in growth are observed between the wildtype and ΔycaO-tfuA strains. In particular, the knockout strain grows more slowly on DMS and acetate, and more slowly on methanol if the temperature is raised. From these growth data, the authors suggest that the thioglycine modification does not affect MCR catalysis or substrate affinity, but instead increases its thermal stability. Although this model is consistent with the results, biochemical characterization of the MCR is essential to addressing this hypothesis. In addition, forming a thioamide seems like a metabolically expensive way to increase thermostability as many other proteins are thermostable without such post-translational modifications. The authors note that biochemical work is beyond the scope of this study, but I am not sure that the knockout alone merits publication in eLife; it seems more suited to a specialized journal.

As noted by other reviewers, and in the letter from the Editor, biochemical characterization of MCR is truly beyond the scope of this manuscript. During the purification process, the Ni(I) in F430 is especially prone to oxidation to Ni(II) and reductive activation of the enzyme in vitro is especially challenging and has only been demonstrated for the MCR from a different, distantly related organism (Methanothermobacter marburgensis; Prakash et al. J. Bac 2014). To circumvent this issue, MCR in the cells is oxidized to the stable Ni(III) form in vivo prior to purification, and Ni(III) is reduced to Ni(I) in vitro by the addition of titanium citrate as a reductant. Protocols for the conversion of MCR to the Ni(III) form vary significantly across different methanogenic strains and a robust protocol for M. acetivorans has not been developed yet. We hope to conduct such studies in the future and with this paper we have the motivation to do so.

In terms of thinking about the modification and its potential role in thermal stability or other aspects of function, it would be helpful to have a picture of the MCR active site showing where the glycine residue is located and what interactions it has with other residues.

We have added a new figure supplement (Figure 1—figure supplement 1) to depict the location of the modified thioglycine residue relative to coenzyme B, coenzyme M, F430, as well as the interaction that it has with other residues in the vicinity.

Reviewer #3:

Metcalf et al. address interesting questions regarding post-translational modifications of the last enzyme of the methanogenic pathway in archaea. It has been known for almost two decades that some residues in the active site of the methyl-coenzyme M reductase (Mcr) are modified. Of particular interest is the thioamidation of glycine. The goal was to identify genes encoding enzymes responsible for this modification. The authors build a case for the analysis of two genes (ycaO, tfuA) that tracked in some cases immediately upstream to the Mcr operon. The authors use Methanosarcina acetivorans to perform phenotypic analyses of strains with deletions of the genes of interest. The observed growth phenotypes are explained on the basis of the entry point of different substrates into the methanogenic pathway. The results are consistent with the explanations provided. However, the authors do not validate the presumed biochemical activities of YcaO or TfuA, hence leading to much speculation. The effects reported here could be indirectly leading to the absence of thioamidated glycine. The authors start the Discussion section by stating '[…] these genes (ycaO, tfuA) are directly involved in the post-translational modification of McrA'; I disagree. To support such a statement the authors must show that YcaO/TfuA can modify McrA in vitro.

We agree with the reviewer that in vitro experiments would need to be done to prove that YcaO/TfuA directly modify McrA. Thus, we have modified the first sentence of the Discussion section to soften our stance on the direct involvement of TfuA/YcaO in the post-translational installation of thioglycine. However, we have added additional data showing that both proteins are required in vivo. To do this, we have generated single mutants lacking either ycaO or tfuA and conducted MS analyses of McrA from each of these strains. Our results (subsection “The ∆ycaO-tfuA mutant lacks the McrA Gly465 thioamide” and Figure 4) indicate that both genes seem to be required for the installation of the thioamide bond.

Reviewer #4:

The manuscript by Metcalf et al. for eLife describes the important finding that the post-translational installation of thioglycine in methyl-coenzyme M reductase in the methanogenic archaeon Methanosarcina acetivorans is mediated by the tfuA-ycaO locus. This experimental procedure showing this is perfect and the results obtained convincing. The excellent manuscript should be published after considering the following minor points:

Discussion paragraph three and following: Yes, MCR is generally thought to be the rate limiting enzyme in methanogenesis, however, the only step in methanogenesis that is physiologically irreversible, is the heterodisulfide reductase reaction rather than the MCR reaction. Thus MCR is most probably not rate limiting. This should be considered or at least discussed.

We have addressed the reviewer’s comment in the Discussion section.

Of the many glycins in McrA only one is converted to a thioglycin and this glycine is buried deep within the McrABC complex. The post-translational modification must therefore occur as long as the protein is not yet folded. Generally enzymes mediating post-translational modifications specifically recognize the sequence upstream and downstream of the amino acid to be modified. Could the authors comment on this in the manuscript with a few sentences?

We have briefly discussed the putative roles of TfuA, including being involved in sequence recognition in the Discussion.

[Editors' note: the author responses to the re-review follow.]

The consensus is that in the absence of supporting biochemical data, the manuscript still pushes a mechanistic message too strongly. Please moderate the discussion. Your results have shown that the thioglycine within the active site is not essential for full activity, not that it lacks a direct catalytic role. In addition, the paragraph on the discussion of growth phenotypes is convoluted and overly speculative. It would be better to tighten and moderate this paragraph, in the absence of supporting data. Growth phenotypes should also be mentioned in the Abstract, so it is clear that the phenotype of the double mutant extends beyond the lack of a PTM.

We have modified the Discussion to soften our stance on the mechanistic underpinnings of the thioglycine modification (Discussion, paragraph three). The growth phenotypes are now discussed in the Abstract.

While we agree that additional biochemical experiments are outside the scope of this work, for this manuscript to be published in eLife as a genetics/physiology paper, these aspects need to be airtight. Towards that end, please include complementation experiments for the mutants.

As requested by the editor, we have performed complementation experiments for each of the mutants (∆ycao-tfuA, ∆ycaO, and ∆tfuA). Details pertaining to these experiments can be found in the Results section (subsection “The ∆ycaO-tfuA mutant lacks the McrA Gly465 thioamide”) and also in Figure 4 Panels E, F, G.

Author details

1. Dipti D Nayak

   Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8390-7251

2. Nilkamal Mahanta

   Department of Chemistry, University of Illinois, Urbana, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8901-2531

3. Douglas A Mitchell

   1. Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, United States
   2. Department of Chemistry, University of Illinois, Urbana, United States
   3. Department of Microbiology, University of Illinois, Urbana, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   douglasm@illinois.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9564-0953

4. William W Metcalf

   1. Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, United States
   2. Department of Microbiology, University of Illinois, Urbana, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   metcalf@illinois.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0182-0671

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