Inhibition Mechanism of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus by Flavonoids: An Enzyme Kinetic and Structural Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

catalysts-3478678_Response to the authors

The article appears to present a well-structured study investigating the inhibition of Vibrio parahaemolyticus lecithin-dependent hemolysin (vpLDH) by flavonoids. The approach combines enzyme kinetics, fluorescence quenching, and molecular dynamics simulations, which is a strong multi-faceted strategy. It addresses a relevant research question regarding anti-virulence strategies against a pathogenic bacterium. Also, the study explores the potential of natural flavonoids (quercetin, morin, and EGCG) as anti-virulence agents, which is a promising approach to combat antibiotic resistance.

The main criticism relates to the use of Lineweaver-Burk plots for determining inhibition mechanisms and constants. This method is known to distort error distribution and can lead to inaccurate conclusions, especially when working with low substrate concentrations.

#1. Outdated graphical methodology: The study relies heavily on Lineweaver-Burk plots for determining inhibition types and constants. This method is considered outdated and prone to errors, especially when dealing with low substrate concentrations.

#2. Limited inhibitor concentrations: The study appears to use only a few inhibitor concentrations, which may not provide a complete picture of the inhibition kinetics.

#3. Fitting: The study doesn't utilize the appropriate methodology for fitting in enzyme kinetics data, e.g. non-linear regression, which could provide more accurate and reliable results.

Suggestions for improvement

Instead of relying on Lineweaver-Burk plots, we would recommend using a non-linear regression analysis of the Michaelis-Menten equation. This approach, combined with global fitting, would provide more accurate and reliable results. Here's a suggested methodology:

1. Collect initial velocity data for a range of substrate concentrations (ideally, from 0.2 Km to 5 Km) at several fixed inhibitor concentrations.
2. Use non-linear regression software (e.g., GraphPad Prism, DynaFit, or R with the drc package) to fit the data directly to the Michaelis-Menten equation. Excell using the supplement Solver can do NLR also!
3. Compare the fits of different inhibition models (competitive, non-competitive, uncompetitive, mixed) using statistical criteria such as AIC (Akaike Information Criterion) or F-test.
4. Use global fitting to simultaneously analyze all datasets (different inhibitor concentrations) to determine the inhibition mechanism and constants.
5. Report the best-fit values for Km, Vmax, and Ki along with their confidence intervals.
6. Provide residual plots to assess the quality of the fit and identify any systematic deviations.

This approach would provide a more robust and statistically sound analysis of the enzyme inhibition kinetics, leading to more reliable conclusions about the inhibition mechanisms and constants of the flavonoids on vpLDH.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Inhibition mechanism of lecithin-dependent hemolysin from Vibrio parahaemolyticus by flavonoids: An enzyme kinetic and structural approach” has been reviewed. Authors demonstrated flavonoids are inhibitor and its potential as natural anti virulence against this microbe. After evaluation, this manuscript is suitable for its publication on this Journal, and it is recommended to publish with major revision.

Specific comments:

1. Introduction: What is the advantages of these flavonoids? Also, their disadvantages should be provided if they are used as natural antimicrobial. More research concerning about your works should be provided.
2. Results:

SD should be added in all results. Also, letters indicating the significant differences should be added in all results.

Deep in scientific reason of all results should be discussed.

Competitive inhibitor was concluded. Why? Please give more mechanism of each compounds that can inhibit this microb.

Please compare with other previous work in all results.

1. Methods:

STAT should be added.

Experimental design should be provided.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes the characterization of three natural products as inhibitors of a virulence factor of the pathogenic bacteria Vibrio parahaemolyticus: lecithin-dependent hemolysin (vpLDH). The methodology is clear and describes in detail all the procedures performed. The main results validated the inhibition of the enzyme vpLDH by three natural molecules using two methods. Besides, they proposed a binding mode combining the inhibition modality identified by the kinetics assays and molecular dynamic simulations. This work makes relevant contributions to the potential of natural products in developing antivirulence agents against Vibrio parahaemolyticus.

However, I have some concerns about the manuscript.

Major issues

In Figure 1 the values of the Ki are in mM but through the manuscript they are in μM. At first glance you could think it is a typesetting error, however, the units in the secondary plots of Figure 1 (slope vs [Inhibitor]) are also in mM so the units of Ki should be in the same units. This is of great importance because depending on the units the Ki values (determined from the kinetics assays) and the K_d (obtained from the fluorescence assays) values are similar or not. Could you clarify this difference in Ki units?

I also recommend a global fitting of the inhibition kinetics data to the Michaelis-Menten equation for the different inhibition models, instead of transforming the data to fit it into the Lineweaver-Burk (LB) equation. For instance, on the LB plots for EGCG and Morin, the lines seem to intersect outside the y-axis, which means their inhibition modality is not competitive and both parameters (Vmax and Km) change in the inhibitors' presence. This could explain the distribution of points in the secondary plots for these inhibitors

Minor issues

On page 5 line 160 “other studies have focused on LDH as a.  molecular target for inhibition by metal ions and chemical compounds” Natural products are also chemical compounds, so maybe the authors intended to emphasize the difference between synthetic and natural products.

The buffer used during the refolding of vpLDH is not specified.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

We appreciate the authors' efforts in responding to our suggestions to improve the manuscript in the enzymatic kinetics part.

Reviewer 2 Report

Comments and Suggestions for Authors

All the points raised have been addressed, and the amended manuscript is now suitable for acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

All my concerns about the manuscript have been solved.