Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii
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Methanocaldococcus jannaschii ( Mj ), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F 420 -dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F 420 (F 420 H 2 ) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that Mj Fsr reduced nitrite to ammonia with F 420 H 2 with physiologically relevant K m values (nitrite, 8.9 µM; F 420 H 2 , 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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Comments to Author
Please accept without further revision.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
In the future, I would recommend at least putting in a sentence that a bioinformatic search for other nitrite reductases returned nothing rather then not mentioning it at all. Overall glad that all comments and suggestions were addressed.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human …
Comments to Author
In the future, I would recommend at least putting in a sentence that a bioinformatic search for other nitrite reductases returned nothing rather then not mentioning it at all. Overall glad that all comments and suggestions were addressed.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments. Dear Dr Biswarup Mukhopadhyay, Thank you for your submission, i would like to apologies on the delay, it took quite some time to acquire appropriate reviews for the manuscript. The reviews have generally positive views for the manuscript and I've selected minor amendments. Please address the reviewers comments, there is only one call for additional experimentation and one of the options requested is only bioinformatic. Best wishes, John.
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Comments to Author
The manuscript by Heryakusuma et al describes a kinetic analysis of nitrite reductase activity of the F420-dependent sulfite reductase from Methanocaldococcus jannaschii. The report is brief, involving confirmation that the enzyme behaves as previously reported, along with the determination of Km/Vmax values for nitrite and FADH2. The likely intermediate, hydroxylamine, is also studied. The data indicate that the enzyme is as effective in reducing nitrite as it is in reducing sulfite, leading the authors to suggest that this may be physiologically relevant. The study is scientifically sound. There are some points (detailed below) that should be addressed by the authors in preparing a revised version. The impact statement is not very clear. There is mention of an infinite supply of nitrite but at low …
Comments to Author
The manuscript by Heryakusuma et al describes a kinetic analysis of nitrite reductase activity of the F420-dependent sulfite reductase from Methanocaldococcus jannaschii. The report is brief, involving confirmation that the enzyme behaves as previously reported, along with the determination of Km/Vmax values for nitrite and FADH2. The likely intermediate, hydroxylamine, is also studied. The data indicate that the enzyme is as effective in reducing nitrite as it is in reducing sulfite, leading the authors to suggest that this may be physiologically relevant. The study is scientifically sound. There are some points (detailed below) that should be addressed by the authors in preparing a revised version. The impact statement is not very clear. There is mention of an infinite supply of nitrite but at low concentration. I am not sure what point is being made with the 'infinite supply' statement - is there a difference here compared to any other environmental chemical component? Also, there is no mention of the toxicity of nitrite that would require a detoxification system. Methods Although it is recognised that it's not necessary to repeat previously published methods in detail, it is also not really acceptable to repeatedly state that methods used were as previously published. The reader of this paper currently gets very little information about the methods employed without consulting previous literature. Please provide outline methods at least. Results L115 'The purified enzyme displayed a UV-visible spectrum typical of siroheme in low-spin ferric state with peaks at 280, 390, 117 and 590 nm (Fig. 1B) (14, 30)'. The enzyme also contains 6 [4Fe-4S] clusters, which each contribute to the absorbance in the near UV/visible region. Is the Fe-S cluster contribution to the absorbance spectrum known? Presumably the absorption due to FAD is difficult to see amongst the siroheme/Fe-S cluster absorbance? Figure 1C Perhaps I misunderstand what this is, but why is the FAD standard elution time shifted from that of the co-injection sample (and sample extract)? L150 From an average of the data from the estimation of products in three independent 30 minutes long FNiR reactions with MjFsrI. This sentence does not make sense; it is lacking a verb. L151 From the data presented, the 3:1 ratio for FADH2 consumed to ammonia produced is what would be expected for the six electron reduction of nitrite. How was the ammonia production determined (I did not see this in the methods)? Also, a rough calculation indicated that the enzyme should be capable of turning over all of the substrate added in 30 min, but it doesn't - only about half of the FADH2 is consumed (limiting substrate). Why is this? I note that the authors in the conclusion section suggest that ~100% of the FADH2 is utilised in the reaction, but 0.043/0.08 is ~53%. The discussion section argues that the nitrite reductase activity of MjFsrI could be physiologically relevant, both for detoxification of nitrite and as a source of nitrogen. There is a rough calculation that supports this based on levels of nitrite and enzyme. In the introduction it is mentioned that several Methanocaldococcus species utilize nitrate as nitrogen source. Does M. jannaschii have this capability? The first step in this process is the reduction of nitrate to nitrite, so nitrite is generated endogenously in such organisms. If this occurs here, then there is all the more reason to need a nitrite reductase. On that note, the authors need to provide further information - what other potential nitrite reductases are present in M. jannaschii?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The authors set up the background that Methanocaldococcus jannaschii is a well studied methanogen. They also make the case that in Mj native habitat, there is a low level but consistent supply of oxidized nitrogen compounds, Finally, they present the premise that an abundant protein Mj-FsrI, previously shown to be a sulfitre reductase, can also act as a nitrite reductase. In fact, this multi- substrate function has been demonstrated with many habitually similar organisms. Excellent experimental setup and rationale for the methods used 2. Presentation of results The results were laid out well. There appears to be another band that runs between 100 kDa and 150 kDa; any speculation about what this band is? It could have …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data The authors set up the background that Methanocaldococcus jannaschii is a well studied methanogen. They also make the case that in Mj native habitat, there is a low level but consistent supply of oxidized nitrogen compounds, Finally, they present the premise that an abundant protein Mj-FsrI, previously shown to be a sulfitre reductase, can also act as a nitrite reductase. In fact, this multi- substrate function has been demonstrated with many habitually similar organisms. Excellent experimental setup and rationale for the methods used 2. Presentation of results The results were laid out well. There appears to be another band that runs between 100 kDa and 150 kDa; any speculation about what this band is? It could have benefited from Mass Spec on the isolated protein to validate that was the target, if MjFsrI specific antibodies are non-existant; however, the explanation was thorough enough to be convincing . 3. How the style and organization of the paper communicates and represents key findings The paper is organized well. It is compact and takes the reader from point to point, culminating in MjFsrI has nitrite reduction capability and it could be an advantage in its native habitat. There is no filler in this paper. 4. Literature analysis or discussion The discussion is done well and delves deeply into the estimates of how the enzymatic reduction of nitrite by MjFsrI could be essential for growth in the native habitat. The estimates of the nitrite assimilation from environmental concentrations through cell uptake and enzymatic conversion were compelling. Given how well the bulk of the discussion went, the last sentence (Line 242-243) seemed like an after thought. 5. Any other relevant comments Lines 44-50: Sets up the premise that nitrate/nitrite/hydroxylamine is broadly toxic to methanogens, then says there are pathways certain methanogens utilize to detoxify, or even benefit from, these substrates. Could the premise be that nitrate/nitrite/hydoxylamine processing occurs in a subset of methanogenic organisms which benefit from nitrite? Line 51-57: Similar argument to Lines 44-50. Line 120-121: If the determination that MjFsrI-flavin is FAD, why does the HPLC elution peak in Figure 1C not line up with the purified MjFsrI-flavin ? Line 238: italicize "in situ"
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
Major: The manuscript by Heryakusuma et al. describes the ability of an F420-dependent sulfite reductase (Fsr) from M. jannaschii to reduce nitrite to ammonium, which was supported by a nitrite reductase activity assay. Based on this finding, the authors predict that the M. jannaschii Fsr may be involved in nitrite detoxification given that the environment where the organism thrives has an infinite availability of this oxyanion. It is widely known that sulfite reductases, as well as nitrite reductases, catalyze both sulfite and nitrite, and this is primarily due to the physicochemical similarities of the two substrates. Given this fact, it does not necessarily follow that a sulfite reductase may be involved in nitrite detoxification or that a nitrite reductase is involved in sulfite detoxification. …
Comments to Author
Major: The manuscript by Heryakusuma et al. describes the ability of an F420-dependent sulfite reductase (Fsr) from M. jannaschii to reduce nitrite to ammonium, which was supported by a nitrite reductase activity assay. Based on this finding, the authors predict that the M. jannaschii Fsr may be involved in nitrite detoxification given that the environment where the organism thrives has an infinite availability of this oxyanion. It is widely known that sulfite reductases, as well as nitrite reductases, catalyze both sulfite and nitrite, and this is primarily due to the physicochemical similarities of the two substrates. Given this fact, it does not necessarily follow that a sulfite reductase may be involved in nitrite detoxification or that a nitrite reductase is involved in sulfite detoxification. Therefore, the claim that M. jannaschii Fsr may be involved in nitrite detoxification must be sufficiently substantiated. One way to do this is to perform RT-qPCR to determine the level of transcription of the fsr gene in the presence vs absence of nitrite or prove by using bioinformatics that M. jannaschii does not possess a homolog of any known nitrite reductases. I recommend accepting this manuscript if one of the two suggested experiments is accomplished. Minor: the authors may apply suggestions 1-3 in their future work. 1) The degradation seen in SDS-PAGE is likely from proteolysis given that the authors did not use any protease inhibitors during enzyme purification. The addition of a protease inhibitor (e.g., Roche Complete, Mini Protease Inhibitor Cocktail) especially during cell lysis may prevent this degradation. 2) Colorimetric methods that are used to quantify proteins are not accurate. This is because the binding behavior of the dyes used in these assays towards the protein of interest may be different from that of the standard protein. Since M. jannaschii Fsr contains Fe ions, an ICP-OES experiment is more appropriate for this quantification - Virginia Tech ICP facility can be found here: https://www.soiltest.vt.edu/Files/labinfo.html. For enzymes that do not harbor metal ions, amino acid analysis is more appropriate. 3) I would like to encourage the authors to provide the full details of the procedure within the manuscript instead of redirecting the readers to the reference articles from which the procedures were followed. This practice will be more convenient for the readers. 4) In Figure 1D, the spectrum for M. jannaschii FAD should be labeled as MjFsrI FAD. 5) The sentence, "From an average of the data from the estimation of products in three150 independent 30 minutes long FNiR reactions with MjFsrI" appears to be out of place.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes