PONE-D-24-55837Investigating the quaternary structure of a homomultimeric catechol 1,2-dioxygenase: An integrative structural biology study.PLOS ONE

Dear Dr. Rudiño-Piñera,

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[The investigation presented here was funded by the Dirección General de Asuntos del Personal Académico (DGAPA) at the Universidad Nacional Autónoma de México (UNAM) through PAPIIT grants IN226523 and IG200223. This research utilized resources from the 16-ID LIX beamline of the National Synchrotron Light Source II, a user facility managed by the U.S. Department of Energy (DOE) Office of Science, which the Brookhaven National Laboratory operates under Contract No. DE-SC0012704. Arisbeth Guadalupe Almeida-Juarez was supported by a doctoral scholarship (2019-000002-01NACF-12588) granted by CONAHCyT, Mexico, and by the incentive as a member of the program Ayudantes de Investigador nivel III provided by the SNI. Enrique Rudiño-Piñera gratefully acknowledges financial support from the institutional budget from Instituto de Biotecnología , UNAM, and the economic incentive from Sistema Nacional de Investigadoras e Investigadores (SNI), México].

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The present study reports novel oligomeric forms of a catechol dioxygenase, an enzyme that is generally regarded as a dimeric protein.

Protein chemists are aware that proteins can form aggregates and aggregation is usually associated with (partial) unfolding, particle formation and loss of activity.

Here it is shown that before large aggregates form, a dimeric protein can form distinct tetramers and octamers, in addition to larger oligomers. The catechol dioxygenase tetramers retained full activity, demonstrating that an enzyme that retained activity after storage could nevertheless have been changed by the storage procedure. Additional quality control by light scattering or analytical gelfiltration would be advisable to control the storage procedure.

The present manuscript contains useful data, however the presentation of the results and the discussion should be improved. It remained unclear why the enzyme was lyophilized in the first place. The general problem of quality control of stored proteins should be discussed in more detail. The importance of the present study for bioremediation should also be outlined much more clearly.

Except for CD and PAGE, the results for the not-lyophilized enzyme are missing. Please add this data for gelfiltration and SAXS.

Minor issues

1. Abstract, “All these characteristics make SfC12DO a very promising candidate for extensive bioremediation applications in polluted soils or waters. “ – why? please elaborate.

2. The authors state, “All relevant data are within the manuscript and its Supporting Information files.” , however I could not find any raw data. Please add excel or csv files with raw data. Add PDB files of the models as supplementary information.

3. Difference between SfsC12DO, Sf12DO and SfC12DO?

4. page 3 line 44, the β is missing

5. Introduction: please add the alternative name 1,2-dihydroxybenzene for catechol

6. Merk → Merck

7. correction of “sample types-lyophilized-reconstituted”

8. ε = 16.8 mM, ε = 16.8 M, seems wrong and these are wrong units for an absorption coefficient (M-1 cm-1).

9. Figure 2, secondary structure cannot be predicted with ESpript, this software just displays it.

10. Figure 3, text font too small, peaks should be labelled p1, p2…, please include elution volumes of SfC12DO elution peaks in the inset (logMW vs. Ve/Vo). Please add a gelfiltration chromatogram of the enzyme which was not lyophilized for comparison. There isn’t a reference to Fig 2A in the main text, only 2B and 2C are referenced. 2C, RMSD compared to what?

11. Correction of “The CD analysis of SfC12DO's showed”

12. “non-lyophilized protein (15 ± 0.25 U/mg).” If the value is 15.00 then change to 15.00 ± 0.25 U/mg

13. Figure 5, It is unclear which panel shows which elution peak and what the diagrams actually show. If this are experimental data, where are the theoretical curves corresponding to the models? Please redo this figure and legend so that it becomes understandable for readers who are not familiar with the SAXS technique. SAXS data of the not-lyophilized enzyme would be needed.

14. Fig S3, what are the colors , what is shown here?

15. Fig. 6, there is no panel D. Panel C, I do not understand what is shown here at all.

16. “In contrast, diffusion problems might prevent it” – it = the substrate?

Reviewer #2: The authors describe the multimeric states of a catechol dehydrogenase enzyme and its impact on its activity. Overall, the manuscript is well laid out. I have a few recommendations for the authors and a few clarifications for the methods.

1. in Figure 2C, the authors have a table explaining the structural nuances for the few studied C12DOs; this would be improved for the reader if they could add the structures and their comparisons for domains and catalytic sites that would convey much more effectively.

2. What is the buffer for the enzymatic activity assay? Does the assay performance change for the Lyo and non-lyo material in different salinity?

3. For Figure 3B, can the authors add both the native and denaturing gel bands? Also, why not have all four peaks on the gel? They're tentatively in the right MW size range to run a native gel for all four peaks.

4. There definitely needs to be an overlay of the SEC profile for the non-lyo and lyo material to make that clear these multimers reflect the nature of the material.

5. IF there are gel lanes for all four peaks, then the enzymatic assay from those fractions is a good continuation.

I think the authors making these edits will improve the overall manuscript.

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Reviewer #1: No

Reviewer #2: No

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Investigating the quaternary structure of a homomultimeric catechol 1,2-dioxygenase: An integrative structural biology study.

PONE-D-24-55837R1

Dear Prof. Enrique Rudiño-Piñera,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have significantly improved the manuscript according to the referee reports. I would recommend to publish the paper now.

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

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