PONE-D-22-14060Structure of Klebsiella Pneumoniae Adenosine Monophosphate NucleosidasePLOS ONE

Dear Dr. French,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Manuscript has been seen by 4 reviewers, two with cryoEM expertise and two being subject experts. Please address all the concerns.

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Please address all the concerns raised by the reviewers.

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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: Partly

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: I Don't Know

Reviewer #4: 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: No

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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 article reports the first cryo-EM structure of an Amn enzyme except no additional structural information is provided by the authors. The authors did not provide any CryoEM micrographs, images, 2D classification images or 3D reconstruction details.

As described by authors the EM structure is not providing any new information regarding the KpAmn structure only physiologically irrelevant different loop conformations due crystal contacts in the crystal structure.

The authors either rewrite the manuscript in terms of methodology to describe Cryo-EM technique.

Reviewer #2: This paper by Richardson et al. describes the cryo- EM structure of adenosine monophosphate nucleosidase (Amn) from a pathogenic bacteria, Klebsiella pneumoniae, which causes hospital-acquired pneumonia and sepsis. Here are the points for revision.

Major points

This manuscript suffers from poor data analysis, the author says in the result, “The cryo-EM structure of K. pneumoniae Amn primarily differs from the apo homologs in its loop regions away from the active site (Figure 4A), either by lacking density for the loop entirely or being in a clearly different conformation”

-In case of lacking density, how the model was built for this region? I do not see any disconnect, missing segment, in Figure 4a.

Figure 4a does not reflect differences clearly, and a magnified view of the loop region would be needed to see the actual conformation differences.

At 3.1 Å resolution, the difference for side-chain residues should be clear. A picture showing side-chain confirmation would be helpful to reach this conclusion.

In catalytic activity, the Km and Vmax for the other two species were performed in different experiments, not at the same time and in the same setup. Reaching a conclusion may not be appropriate. Some unnecessary statement “though the --------- intervening decades”, is not required.

In figure 2B, I don’t see high-resolution features such as density for bulky side chain residues, particularly in a helix. That should be evident at this 3.1 Å resolution, and it looks overall like a low-resolution map. To gain readers’ confidence, detailed cryo-EM data processing information, including representative micrographs, 2D class, 3D class, and final post processed is required.

Minor point

In introduction; The acronym ESKAPE in the introduction needs expansion.

In result; the Figure 2 parts are not in order e.g. Figure 2C comes first in the text.

In result; ‘mixed secondary structure’ is not correct, needs to explain in terms of the secondary structure topology or domain. Same in Figure 2 legend also.

1T8S is a PDB id, it should be written as PDB ID; 1T8S, throughout the text.

In table 1; add one more column for the name of the organism for the source of the protein.

On searching EMD ID 26838 in EMDB shows no entry. Check this out.

Reviewer #3: The manuscript describes the structure of K. pneumoniae adenosine monophosphate nucleosidase determined by cryoelectron

microscopy and structure is compared with other known structures.

Overall manuscript is informative bur there are several concerns. The overall structure of the enzyme is not described, at least it should have been described briefly.

There are no images of EM, secondary classification, other than Figure 2B image. At least these should have been in supplementary data.

The authors describe structural differences in loops at such a low resolution. Are the lops well defines in EM structure?

Can they use this low resolution structure for drug design or inhibitor screening?

The manuscript does not describe or give the reference to the literature, explaining the importance of this enzyme. Does the importance of this enzyme shown in this organism?

Overall the authors should describe the manuscript with more information.

Reviewer #4: This paper reports the cryo-EM structure and catalytic activity of Adenosine Monophosphate Nucleosidase from Klebsiella Pneumoniae. However, the mauscript should be revised considering the following comments.

Comment 1: Page3

“Biochemical studies of nucleotide processing pathways in Azotobacter vinelandii and

Escherichia coli (E. coli) identified a key difference between prokaryotes and eukaryotes in their

regulation of the levels of adenosine monophosphate (AMP), a key component of both RNA

synthesis and energy storage.” Provide References.

Comment 2: Page 8

All PDB structures identified by BLAST as

close Amn homologs were aligned to Amn in UCSF Chimera (A)

Provide sequence comparison highlighting the conserved residues and discuss functionally significant residues.

Comment 3:

Page 8 line number 145:

“broader comparison of K. pneumoniae Amn to its homologs can be assessed by

sequence conservation”

Provide the phylogenetic relation between the available structures.

Comment 4:

Page 8 line number 149:

“the loop covering the active site is strongly conserved”

Provide the structural alignment of the active site residues.

Comment 5:

Page 10 line number 182

“while the crystal structure leads to significant underestimation of the flexibility of several N-terminal loops the catalytic core of the enzyme is effectively invariant between these species.”

Discuss any functional significance of the N-terminal loops in Amn, if not then there is no need to include this sentence.

Comment 6:

The discussion should be precise and elaborate and should only discuss their findings. The KpAmn structure should be compared with that of the other homologs in detail highlighting the functionally relevant substructures.

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Reviewer #1: Yes: Dr. Ethayathulla Abdul samath

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Pravindra Kumar

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Attachments

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Submitted filename: review.docx

Structure of Klebsiella Pneumoniae Adenosine Monophosphate Nucleosidase

PONE-D-22-14060R1

Dear Dr. French,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Janesh Kumar, Oh.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please remove the older version of Fig. 3 and provide PDB/EMDB deposition details in the final version before publication.

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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: 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 #2: No

Reviewer #3: Yes

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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 #2: The authors have addressed all the points except not shared PDB ID and EMDB ID. Please make sure the coordinate and Cryo-EM maps are available for the scientific community immediately after publication.

Reviewer #3: Almost all of our questions and observations were addressed. The present version of the manuscript can be accepted.

Reviewer #4: Authors have submitted the revised manuscript on "Structure of Klebsiella Pneumoniae Adenosine Monophosphate Nucleosidase". They have answered all the queries and comments made by me.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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 #2: No

Reviewer #3: No

Reviewer #4: No

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