The Impact of Bilirubin on 7α- and 7β-Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

All the issues raised in the review process have been addressed correctly and properly.

Author Response

Thanks very much for your review.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript „The impact of bilirubin on 7α- and 7β- hydroxysteroid dehydrogenases: Spectra and docking analysis” by Qingzhi Ji et al. presents the study on effects of bilirubin on enzymatic activity of two hydroxysteroid dehydrogenases.

The main question concerning this manuscript results from the last sentence of the abstract:

This research not only provides the more in-depth understanding of 7α- and 7β-HSDH, but also reminds us to avoid the inhibition of bilirubin on hydroxysteroid dehydrogenases.

First of all, effects of bilirubin on HSDH were already studied by this research group (i.e. references 16 and 22) and the new material should be clearly marked. The description in lines 91-102 is not sufficient, as some information mentioned as new was already presented (HPLC, docking). Figure S2 is practically identical to the one presented in reference 22 (figure caption differes in visualization software).
Even more important question concerns the reason to study this interaction. The Authors mention that bilirubin is one of the main components of waste chicken bile, from which substrate TCDCA is obtained (line 59) and the idea of using chicken bile as a source for important material is interesting (however, also already published). There is no information on the concentration of bilirubin in chicken bile or ratio to TCDCA in this work, or possible methods to avoid bilirubin inhibition – or any possible benefits from this inhibition. The information in lines 185-189 needs more comments.

 

Therefore, the message from this article is not clear and its importance is not sufficiently presented.

 

Specific questions:

Please explain the clean process in the sentence (line 61) in relation to this text. “In summary, this strategy affords an efficient and clean process for production of TUDCA”.

Please provide reference for the sentence “Recently, bilirubin has been recognized as a hormone with endocrine actions.” (line 66) In general, what is the importance of the biological activity of bilirubin in the context of its inhibitory activity? Do the Authors plan to suggest the use of product mixture?

Throughout the text, Authors use “combination” instead of reaction, formation, synthesis, both covalent and non-covalent (compare the use in line 76 and 77), the combinations of bilirubin to enzymes (line 255). In my opinion, a more specific vocabulary should be used.

I would suggest using UV, fluorescence and CD spectroscopy as mentioned methods, spectra are results of measurements performed using these methods.

The SDS-PAGE results with a band at track 1 suggest mass below 26 kD, please check the text and Figure 2.

Please provide reference for Peptide calculator (line 117) and information on sequence of HSDH in results, in the text, not in Table 1 footnote. The reference to conditions of enzyme activity tests (par. 2.2) should be also mentioned as the experimental part is at the end of the text.

Please explain why the charge of HSDH is presented in mV (Both 7.- and 7ß-HSDH are negatively charged at pH 7.0, -2.8 mV for 7.-HSDH and -4.6 mV for 7ß-HSDH).

Some conclusions are presented much earlier than the respective data (for example lines 137, 138: “Due to different amino acid compositions and three-dimensional structures, there are differences in the maximum binding amount of bilirubin to 7.- or 7ß-HSDH.)

In some cases, the units may need verification or more information (line 87: concentration of about 25 nM/mg), line 396 and 398 for bilirubin (micromolar or milimolar?), line 49: moL/L

Figure 3: the scale on X axis is out of proportion.

There is no information on control measurements, especially effect of bilirubin on NADP+.

The pH of enzymatic tests (8.5) is different than pH used in spectral studies (7.4). Please explain this difference and possible effects on observed changes.

Please explain (line 240): The different results may be attributed to different compositions of tryptophan and tyrosine.

Did the Authors analyze CD spectra from 200 to 300 nm for protein secondary structure? The conclusions on the structure changes without this part are not complete. How was the concentration change compensated in CD analysis with different ratios of HSDH and bilirubin?

There are well explained concepts in the text, therefore the competitive and noncompetitive inhibition (line 474) would benefit from clear indication in the results (line 311), as competitive kinetic experiments were not discussed sufficiently (or I did not notice this aspect).

The information in lines 185-187 needs comments.

 

Comments on the Quality of English Language

The language of the manuscript needs careful correction (passive voice without main verb, some sentences seem unfinished (line 55/56), order of words (the binding of bilirubin and  albumin has long been explored by scientists, which possess a variety of physiological  functions in human body, Serum unbound bilirubin concentration can act)

In some sentences, the words used do not make much sense or should be replaced with the correct terms.

The fragment (lines 357-361) is rather an instruction, not description.

Line 41: Due to the function of blood–brain barrier crossing

Line 85: that C1 esterase activity in inhibited by unconjugated bilirubin

Line 158: catalysis rate of enzymes became slowly with the inhibition

Line 234: polarity around the chromospheres molecule

Line 311: Refer to the kinetic constants (Table 2), after binding with bilirubin,

Line 313: has a significantly increase.

Line 334: Binding energy is include van der Waals force,

Line 417: Perkinelmer -PerkinElmer

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The changes introduced by Authors and the responses provided by them are sufficient, although there are some questions remaining.

The information in answer 1(4)
"The concentration of bilirubin in chicken bile or ratio to TCDCA is not the center of research"
is surprising taking into account one of the aims of this work (utilization of chicken bile as a source of bile acids for transformation.

Response 6: If the molecular weight of enzymes has been confirmed using other methods than SDS-PAGE, please provide this information.

Please check the X-axis scale in Figure 3. The distances between 0, 0.05, 1 and 2 are identical, making the distance between 0 and 1 twice as long than between 1 and 2. (see 0.4 and 0.8 for the same effect).

Comments on the Quality of English Language

Some minor errors remain

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

   

7Alpha- and 7beta-hydroxysteroid dehydrogenases (HSDHs) catalyse the conversion of taurochenodeoxycholic acid (TCDCA) into tauroursodeoxycholic acid (TUDCA) and in the submitted manuscript the authors report the impact of bilirubin on HSDHs activity. This biotransformation has been reported in references 17, 18 and 23 (some of the authors of the submitted manuscript are among the authors of references 18 and 23). Moreover, in reference 23 it has already been described that the activity of HSDHs is significantly inhibited by bilirubin and a molecular docking study has also been provided (as detailed in sections 2.5, 2.7, 3.4 and 3.5 of ref. 23). Due to these antecedents, the reported work is of very limited novelty.

In addition to these comments, there are some issues to be mentioned:

- In Figure 1, the hydroxy group at C3 in both TCDCA and TUDCA is to be written bearing its alpha stereochemistry.

- Instead of “detection” and “detect” (used through the manuscript), “analysis” and “analyse” should be employed.

- It should be important to add in the Introduction that bilirubin is one of the main components of waste chicken bile, from which substrate TCDCA is obtained (lines 57-58).

- SDS-PAGE is a widely known technique, so that the explanation provided on lines 95-99 should be removed.

- References 20 and 31 are the same.

- The English of the manuscript must be corrected and revised. The following fragments are provided as samples:

“This research was focus…” (line 77): was focused.

“… results may attribute…” (line 210): may be attributed.

“…bilirubin may be bind with…” (line 261): may be bound.

“respond” (line 407): response

“In previous study, we found that the activities of 7α- and 7β-HSDH effected by bilirubin.” (lines 72-73): change “effected” by “affected”.

“The binding of bilirubin and albumin has long been explored by scientists, and which possess a variety of physiological functions…” (lines 67-68): remove “and” in “and which possess”

“… but also reminds us to avoid the effects of bilirubin in the preparation of TUDCA.” (Abstract): confusing statement

- Minor comments:

The names of TUDCA and TCDCA are not provided by their first mention in the Introduction.

E coli (line 88) is not written in italics.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper describes the isolation and purification of two enzymes (7α- and 7β-HSDH), as well as the study of their activity within the frame of their use as biocatalysts. In details, the effects of bilirubin over the catalytic performances of these two enzymes were studied by various methods (UV-Vis, CD, fluorescence, …), leading to the conclusion that the presence of such a molecule negatively affects their biocatalytic abilities. Finally, the binding pose of bilirubin was proposed.

 

The language throughout the paper is appropriate from a technical viewpoint and reading is not impeded, but significant editing needs to be done.

For instance:

- singular and plural nouns are often misplaced (e.g. lines 15-16: “spectra” should be used instead of “spectrum” in both cases)

- some verbs are not conjugated correctly according to the proper person and some tenses are wrong (e.g. line 49: “Depending” or “Relying” should be used instead of “Dependent”; line 135: “rose” should be used instead of “grown”)

- auxiliary verbs are sometimes missing (e.g. line 18: “was reduced” should be used instead of “reduced”)

- some verbs and adverbs/verbs and nouns are swapped (e.g. line 246: “obvious” should be used instead of “obviously”; lines 140-141: “immobilization” should be used instead of “immobilized”)

- some prepositions are missing (e.g. line 69: “in inhibited” should be used instead of “inhibited”)

A thorough language check is suggested.

 

A number of minor revisions should be applied:

- Introduction paragraph: some acronyms are used but the explicit name has been written only in the abstract (for instance: TUDCA, TCDCA, 7α- and 7β-HSDH, …) and not in the body of the paper. Complete names should be stated at least once before using their abbreviations.

- Lines 23-24: “but also reminds us to avoid the 23 effects of bilirubin in the preparation of TUDCA” should be rephrased

- Lines 44-45: stating the year 2022 is fine, but the name of another journal should not be given explicitly in the article (for uniformity reasons with all other references)

- Figure 1A: the stereochemistry of all carbons should be included in the structures of every compound

- Line 64: “synthesized” instead of “created”

- Line 66: “reverts back to” instead of “reverts to become”

- Line 88: “E. coli” should be written in italics (E. coli)

- Line 88: “a mild condition” needs to be further explained

- Lines 95-99: The paragraph is not necessary and should be removed, since it deals with state-of-the-art information about SDS-page

- Lines 100-107: All information is correct but should not be mixed up. Therefore, the one from experimental data should be kept separated from the ones from the peptide calculator, so as not to confuse the two different contributions

- Line 118: “was more seriously” instead of “was seriously”

- Line 187: “442 nm” instead of “420 nm”

- Lines 204-206: The sentence should be explained more in details

- Line 285: “respectively” should be removed

- Paragraph 3.7: the HPLC wavelength is missing

- Table 3: the energy values should be included in the Table

- Line 412: the titles of figures and tables must be written

 

Two major issues must be resolved.

1) Paragraph 2.6 proposes different binding sites for bilirubin starting from in silico calculations. However, the criteria for the choice among them is not clear.

In the case of 7α-HSDH, the Authors state that the most probable pose is represented by structures 1, 3, 5 and 7, but the other six contributions have sometimes lower energy values. It is not clear why they were excluded altogether.

In the case of 7β-HSDH, the paragraph does not allow to clearly understand which site is considered most likely for binding with bilirubin.

2) The docking studies start from the assumption that the binding site of bilirubin is close to the cofactor region of the two enzymes. However, the nature of the inhibition (competitive, non-competitive, …) has not been determined, making it impossible to either identify the site or demonstrate the conclusions of paragraph 2.6.

Similarly, lines 209-211 should be explained more. Previous references are needed to explain how this interaction may depend on the aminoacidic composition of the proteins, citing the interactions between the two enzymes and other known ligands.