Studies on the Selectivity Mechanism of Wild-Type E. coli Thioesterase ‘TesA and Its Mutants for Medium- and Long-Chain Acyl Substrates

Round 1

Reviewer 1 Report

This is a fascinating work. From my point of view, exceptionally useful to streamline the molecular analysis of any protein-substrate interaction. Not only for the shown case but for any pairs under study. However, it needs major English revisions. The grammar feels weird in most paragraphs. 

 

Line 14. Please verify the correct writing for E. coli throughout the manuscript. 

Line 14. Why is TesA written as 'TesA in the document? What does the apostrophe mean? I have never seen this nomenclature elsewhere.

 Line 14. What does "FA" stand for? 

Line 14. What does "MCFA" stand for? 

Line 15. Please avoid casual writing and contractions, such as "It's" in the document.

Line 20. What does "SNAC" stand for? 

Line 22. What does "MM/PBSA" stand for? 

Line 24. Please use a capital first letter for "Van" in "Van der Waals"

 

Figure 1 is not referenced in the paragraph before its appearance.

 

Line 108. The authors repeat "than the wild type" several times in the paragraph.

 

2.1. Molecular Docking Results. How did the authors determine the tertiary protein structures for the docking? Especially for the mutants

 

Line 167. What exactly is related to the stable conformations? The sentence appears incomplete.

 

Line 283. All those sentences are either incomplete or poorly punctuated. 

 

 

Author Response

Thanks to the reviewer's comments, please see the attachment for our corresponding responses.

Author Response File: Author Response.pdf

Reviewer 2 Report

 

Brief summary

The article of Zhang et al. is a computational study on the thioesterase of E. coli, which is an industrially relevant enzyme. Although the work itself can be an interesting case study and an inspiration for those who wish to exploit similar computational methods as a tool for understanding enzyme mechanisms, I have several concerns with the novelty, significance, presentation and scientific soundness of the manuscript. In my opinion, this article needs careful rewriting to be accepted in Catalysts.

General concerns

1.     Novelty and significance

The investigated mutations of the wild type ‘TesA were choosen from the article of Deng et al. In the original article it was shown that these mutants have an increased affinity towards shorter acyl substrates. The original article contains docking studies and structural explanations. There are new computational results in the manuscript of Zhang et al., however, these results do not add significant novelty to the topic.

2.     Quality of presentation

Although there is no limit on the pages for manuscripts submitted for Catalysts, 28 pages for an article such as this is immensely long. For this reason, the text is neither concise nor comprehensive. Several figures (e.g. Fig. 10 and 11) and most tables could be presented in supplementary material instead of the main article. Font size changes during the article and incorrect figure descriptions (e.g. Fig. 5/b) further hinder the understanding of the manuscript.

3.     Scientific soundness

The text is very hard to follow in its current form, The poor usage of scientific expressions lowers the quality of the manuscript as well. Some examples as follows:

L50: “Block”, this could be called region, part, or even motif, instead of blocks

L55: it is catalytic triad, not “triplets”

Furthermore, the conclusion, the most important part of the article is almost incomprehensable. In a study like this, it should be addressed clearly what the computational results add to already existing experimental results. The manuscript in its current form lacks this entirely.

4.     Extensive English editing is needed

The whole manuscript should be revised as it contains incorrect sentences and expressions, the coherence of the text is low, the level of English language used is not eligible for a scientific article (e.g. abbreviations like “it’s”).

 

Author Response

Thanks to the reviewer's comments, please see the attachment for our corresponding response.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Brief summary

The article of Zhang et al. was improved greatly, in its current form it can be a useful computational study on the thioesterase of E. coli. I am grateful that the authors showed great effort towards the development of the manuscript. The English editing increased the quality of the article noticeably. The current manuscript presents the significance and novelty of the author’s work properly. There are only minor concerns left in the article regarding the quality of writing and explanations, which I listed in the following sections. I believe that after correcting the manuscript, it can fully exploit its potential as an interesting study on computational methods as tools for understanding enzyme mechanisms.

General suggestions

1.     Abstract

As other parts of the manuscript, the abstract was improved greatly by the authors. For further improvement, I suggest to include the aim of the study in the abstract. The authors also list several parts of their findings, however, I suggest to end the abstract with a more concrete conclusion.

2.     Significance to the previous sudies

In the current manuscript, the authors discuss it in more detail, how their work adds to the topic of thioestherases. I also thank the authors the detailed response about this.

However, I find it still confusing in some parts of the manuscript, wether the authors refer to their own results or to the reference. This parts should be made clear to help the understanding of readers:

 

L14: “It has been shown” makes it sound like this sentence refers to the current study, and not the reference. If I am correct and this sentence is about the results of Deng et al., the sentence should start with “It had been shown” or “It was shown previously”.

L84: “found that there were to mutants in ‘TesA”, this part is incorrect, Deng et al produced two variants of the wild-type enzyme by rational design, please revise this part accordingly. It could also be elaborated here or elsewhere, what was the reasoning behind this mutants, how these mutations have an effect on the selectivity.

In this paragraph (L84-94) the authors list many numbers on the activity and selectivity of the wild-type and mutant enzymes. I strongly suggest to outline the differences in the enzymes, because their comparison like this is hard to understand.

L106: “in our study” 

3.     Structural description of ‘TesA

This part (L42-53) could be further improved as well. The conserved regions of the protein are listed in the text, but the level of conversation should be mentioned as well.

The authors should also elaborate more on the mentioned oxygen anion hole here or in the next paragraph, the meaning of this is now unclear.

In the legend of Fig.1., the colouring of the figure should be made clear.

4.     Presentation of results

The presentation of the results was improved by the authors as well, but just as the other parts of the manuscript, this can be improved further as well.

Fig.4.: The authors should consider, wheter the projection of the principal components would be more informative than the landscape.

Fig. 5.: The description of the different panels (a, b, c, …) is missing.

Fig. 6.: The description of the different panels (a, b) is missing.

Fig. 7.: To avoid confusions, the molecules should be regarded as a, b, c… panels.

Chapter 2.9.2. still needs some rewriting, some part are hard to understand or confusing (e.g. L473-474). Previous Tables, Figures and Chapters could be referred to make understanding even easier.

5.     Conclusion

The quality of the conclusion increased severely as well. Minor changes still can be made.

Some sentences (especially in the last paragraph) are extremely long and hard to follow, these should be revised.

The authors presented a great amount of computational work in this study, which will help experimenal researchers. It would be useful to summarize, how the results presented in this article can be exploited to further improve thioesterases.

 

6.     Emphasizing the strength of the work

In my opinion, the strength of this study is how it estabilishes an example for computational studies, with a clear workflow and with general methods that can be used by a wide audience. I think this should be emphasized more in the introduction and in the conclusion.

Furthermore, it would be useful to make a graphical workflow to help readers understand easier the steps and methods used in the study.

Minor comments

L68-73: It is still a bit unclear, if the authors refer to their own study, or to all MD simulations with ACP, the text should be improved according to this

Fig.2.: legend is unclear, description of blue colour is missing entirely; “same structure in red”, it should be described properly, what is the red rectangle referring to

L124: “previous studies have shown” please add references of previous studies

L144: It is confusing how the main text refers to “red box” and “blue box” in the supplementary figure, it is misleading as there are figures in the main text with red and blue boxes as well

L416-417: repetition of “additionally”

L612: “information”

L618: please remove “please add”

From L626 in all of the References: The word “Bibliography” is put in every reference, this should be fixed in the final version

Author Response

Thank you for the reviewer's comments. Please see the attachment for the detailed response

Author Response File: Author Response.pdf