From the journal RSC Chemical Biology Peer review history

22-Jul-2023

Dear Dr Wilkinson:

Manuscript ID: CB-ART-07-2023-000127
TITLE: The catechol moiety of obafluorin is essential for antibacterial activity

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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Reviewer 1

This manuscript describes the role of the catechol moiety of the bacterial Thr-tRNA synthetase inhibitor obafluorin. Obafluorin is a beta-lactone natural product produced by Pseudomonas fluorescens that this team has characterized in previous publications. In the current work, the authors explore the role of the catechol component of the antibiotic and demonstrate using a series of very nice mutasynthesis studies that incorporate catechol analogs that 2,3-dihydroxy benzoic adia is essential for activity. Furthermore, they show that the catechol binds ferric iron and that this complex has significantly reduced MIC levels compared to the nono-coordinated obafluorin against relevant bacteria. Through a series of in vitro and in vivo experiments, the authors show the importance of this catechol in enzyme inhibition and the stability of the beta-lactone warhead. This finding is quite fascinating because a recent crystal structure of the target enzyme with obafluorin shows that this catechol coordinates an active site proximal zinc ion, yet the natural product has little affinity in solution for zinc compared to iron. Therefore, this work shows an unexpected relationship between iron-binding properties that improve activity and the requirement for binding zinc at the enzyme's active site.

I very much enjoyed reading this comprehensive and well-written manuscript. My only comment is that the authors should carefully examine the number of significant figures reported in the IC50 table compared to the sensitivity of the in vitro assay. Otherwise, this is an important contribution to the literature and a comprehensive study.

Reviewer 2

Obafluorin is a natural product compound with unique structure and broad-spectrum antibacterial activity. In this manuscript, Batey et al focused on the function of the catechol group of obafluorin.
Using biosynthetic methods, obafluorin analogues removing one or two phenolic hydroxyl groups from the catechol group are synthesized. The authors found that the intact catechol of obafluorin is required for both antibacterial activity and inhibition of the ThrRS target. The authors also showed that obafluorin interacts weakly with zinc ions in solution, but it selectively forms a 1:1 complex with ferric ion. Binding with ferric ion appears to inhibit the hydrolysis of the lactone ring in obafluorin and increase its potency against sensitive bacteria.
The study is technically sound and the data are concise. Therefore, this reviewer recommends the publication of this manuscript.

Minor point:
The 4th line from the bottom of Page 22: The subscript "2" in "MgCl2" is missing.

Dear Professor Yang,

Many thanks to you and the reviewers for the positive comments regarding our manuscript entitled ‘The catechol moiety of obafluorin is essential for antibacterial activity’ and its acceptance for publication dependent upon appropriate revisions which are noted below. We also found a small number of typographical errors which have been corrected and are clearly visible in the tracked copy of the manuscript.

Reviewer 1:
We thank the reviewer for drawing our attention to the number of significant figures quoted for the tabulated IC50 values and have adjusted these to two significant figures in the tables and throughout the text, in line with those used in previous publications.

Reviewer 2:
The typo regarding the formulae for MgCl2 has been corrected on page 22.

Many thanks for taking the time to consider our manuscript and we hope all points have now been addresed and include all requested details and documents in the re-submission.

We remind you that the manuscript was invited as part of the RSC Fellows collection and look forward to hearing from you in due course.

Yours sincerely,
Barrie Wilkinson

11-Aug-2023

Dear Dr Wilkinson:

Manuscript ID: CB-ART-07-2023-000127.R1
TITLE: The catechol moiety of obafluorin is essential for antibacterial activity

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 2

I recommend the publication of the current version of this manuscript.