PONE-D-25-23633Resolution of physics and deep learning-based protein engineering filters : A case study with a lipasePLOS ONE

Dear Dr. Della Corte,

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PLOS ONE

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D.DC and S.G. were supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R15GM155803. D.DC. and P.D. thank the graduate school iGRASPseed for funding. Part of this study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through funding no. JA 448/8-1 to KEJ. G.F.S. and D.DC.

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D.DC and S.G. were supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R15GM155803. D.DC. and P.D. thank the graduate school

22 iGRASPseed for funding. Part of this study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through funding no. JA 448/8-1 to KEJ. G.F.S. and D.DC. gratefully acknowledge the computing time granted by the JARA-HPC Vergabegremium and VSR commission on the supercomputer JUROPA at Forschungszentrum Jülich.

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D.DC and S.G. were supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R15GM155803. D.DC. and P.D. thank the graduate school iGRASPseed for funding. Part of this study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through funding no. JA 448/8-1 to KEJ. G.F.S. and D.DC.

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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

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

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: No

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5. Review Comments to the Author

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Reviewer #1: This study focuses on evaluating the limitations of enzyme design methods through computation in optimizing enzyme activity against non-natural substrates, specifically using the LipA enzyme from Pseudomonas aeruginosa to hydrolyse the Roche ester substrate. There are some of my inputs that can improve the quality of this article.

1) The title is very general. please be more specific

2) Please specify the selection of H14/M16/L17/H81!

3) Compare the RMSF (or RMSD relative to the crystal structure) in the CONCOORD ensemble vs. MD 100 ns. In this article, CONCOORD is mentioned to “lower the pressure” due to constraints. However, doesn't this make the ensemble too “too constrained”, thus losing millisecond-scale motions that can affect binding?

4) The author does not provide an explanation of the computational methods used, for example, AMBER99SB-ILDN and M06-2X/6-31G. Please clarify!

5) Please specify the solvent conditions in the DFT calculations!

6) In Figure 5, what specific criteria (distance & angle) were used to detect H-bonds in the analysis? Were other thresholds (eg: 3.2 Å) tested to see if the pattern changed?

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Reviewer #1: No

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Resolution of physics and deep learning-based protein engineering filters : A case study with a lipase for industrial substrate hydrolysis

PONE-D-25-23633R1

Dear Dr. Della Corte,

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,

Zeynep Ozdemir

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewer #1: All comments have been addressed.

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

**********

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

Reviewer #1: Yes

**********

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

**********

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 revisions provided can be considered satisfactory. The authors have:

1) Addressed scientific transparency: They are now more open in explaining their methodological approach, assumptions, and limitations, so readers understand the context of the results presented.

2) Provided sufficient definition of controversies: Discrepancies between simulation and experimental results are no longer left "hanging," but rather the causes or possible underlying factors are explained.

3) Acknowledged study limitations: The discussion section now emphasizes the limitations of the computational method (for instance, DFT accuracy, parameterization sensitivity), so the conclusions do not seem too absolute.

4) Avoided overinterpretation: The results are now read more cautiously, emphasizing that the substrate recognition mechanism is influenced not only by cargo distribution at the terminal, but also by other, more complex factors.

With these revisions, it is clear that the authors accommodated reviewer's comments and improved the quality of the manuscript. I am satisfied, and the manuscript is worthy of further consideration for publication.

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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 #1: No

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