Feedback Inhibition of DszC, a Crucial Enzyme for Crude Oil Biodessulfurization
Round 1
Reviewer 1 Report
Neves et al study possible binding modes of inhibitors of monooxygenase DszC using computational methods, molecular docking and molecular dynamic simulation. The manuscript is well written and I do not have major objections.
However, the mechanism of feedback inhibition needs some clarification in the introduction. From Figure 1 it seems that DszC catalyzes a three substrate reaction between DBT, FMNH2 and O2. Assuming that the concentration of the molecular oxygen is not varied we have a two substrate reaction. It is mentioned in several places, and this is important, that the inhibition by 2-HBP and HBPS is noncompetitive regarding DBT substrate. What about FMNH2? It is concluded that many binding modes of inhibitors (e.g on page 12 line 433) may affect proper binding of FMN. This left me with the feeling that 2-HBP and HBPS should be competitive inhibitors for the binding of FMNH2 substrate. Please give a more detailed description of the mechanism of inhibition of DszC, what do we know from the kinetic experiments?
The IC50 values (listed in line 315) should be mentioned already in the introduction.
Minor,
Line 180 – Error!.... What do you mean here?
Line 221 – two ns 200 MD ??
Line 264 – is most?
Line 287 – I could not find Arg338 (and Trp205) in Fig 4.
Line 347 – Same supplementary figures for both binding sites?
Author Response
Neves et al study possible binding modes of inhibitors of monooxygenase DszC using computational methods, molecular docking and molecular dynamic simulation. The manuscript is well written and I do not have major objections.
However, the mechanism of feedback inhibition needs some clarification in the introduction. From Figure 1 it seems that DszC catalyzes a three substrate reaction between DBT, FMNH2 and O2. Assuming that the concentration of the molecular oxygen is not varied we have a two substrate reaction. It is mentioned in several places, and this is important, that the inhibition by 2-HBP and HBPS is noncompetitive regarding DBT substrate. What about FMNH2? It is concluded that many binding modes of inhibitors (e.g on page 12 line 433) may affect proper binding of FMN. This left me with the feeling that 2-HBP and HBPS should be competitive inhibitors for the binding of FMNH2 substrate. Please give a more detailed description of the mechanism of inhibition of DszC, what do we know from the kinetic experiments?
The IC50 values (listed in line 315) should be mentioned already in the introduction.
Reply: We have now discussed the kinetics of DszC in more detail in the Introduction section to make it clear that despite that 2-HBP and HBPS are noncompetitive towards DBT, they can compete with FMN binding; we also introduced relevant IC50 values as requested. In page 3, it can now be read:
“A comprehensive kinetic study by Abin-Fuentes evaluated the dependence in activity of the 4S pathway in resting cell cultures and through in vitro experiments with each enzyme in the pathway.[23] The authors confirmed that the feedback inhibition mechanism of the 4S pathway results from the accumulation of the final product within the biocatalyst at concentrations that can surpass the hundreds of μM. In agreement with other work,[34-36] the DszB and DszC enzymes were identified as the kinetic bottlenecks of the 4S pathway, with a catalytic rate, kcat, of 1.7 and 1.6 min-1 and a catalytic efficiency of 1.3 and 1.1 μM-1·min-1. In addition, dose-response experiments on the DszA-C enzymes with 2-HBP and the 2’-hydroxybiphenyl-2-sulfinate (HBPS) product of DszA showed that DszC is the most severely affected by the feedback inhibition caused by the 2-HBP product of DszB and also by the 2’-hydroxybiphenyl-2-sulfinate (HBPS) product of DszA, with half maximal inhibitory concentration (IC50) of 15 and 50 μM. DszA and DszB are also affected, although to a lesser extent.[23] The sensibility to feedback inhibition follows the order DszC > DszA > DszB. In particular, the inhibition of DszC by 2-HBP and HBPS also fitted that of a noncompetitive kinetic model, with inibition constants, Ki, of 40 and 13.5 μM. As such, the binding of 2-HBP and HBPS should not compete with that of the DBT substrate, although it can still compete with FMN binding.”
Minor revisions
Line 180 – Error!.... What do you mean here?
Reply: We have now rewritten the formatting issue to “The other two DszC:ligand systems considered only the occupation of the binding sites common to 2-HBP and HBPS (V and VI in Table 1)”
Line 221 – two ns 200 MD ??
Reply: We have now rewritten the typo to “We ran two 200 ns MD simulations with different starting conditions…”.
Line 264 – is most?
Reply: We have now rewritten the typo to “… we observe that site V is the one occupied in most simulations…”
Line 287 – I could not find Arg338 (and Trp205) in Fig 4.
Reply: The residues are not represented in the Figure 4 because they do not interact with the ligands through close contacts, but are instead closeby to residues interacting with the ligands (as is the case of Arg338) or interact more weakly with the ligands (as is the case of Trp205). To make this clearer, we have rewritten the corresponding sentences: “Both binding sites also include several residues (Gly347-Leu351 or Pro413-Phe415, sites I and VII in Figure 4) close to Arg338, whose mutation by Ala affected DszC activity and oligomerization.[39] The binding site IV comprises several residues involved in the binding of the FMN cofactor, namely the conserved Phe161 and Trp205, and Arg370, whose mutation by Ala have led to inactive forms of DszC,[38] although Trp205 establishes close contacts less often.
Line 347 – Same supplementary figures for both binding sites?
Reply: Figures S7 to S10 correspond to a comparison of the frequency of contacts for 2-HBP and HBPS ligands by condition of the simulation. In the referred line we state that binding sites II was identified in Figures S7 and S9, whereas site VII was identified in Figures S7 to S9, which means S7, S8 and S9.
Reviewer 2 Report
In this manuscript, the authors identified potential binding sites for substrates of DszC related to crude oil biodessulfurization. The authors also propose the mechanism of feedback inhibition of DszC, based on the results of molecular docking and molecular dynamics simulations. Considering that these results can be applied to protein engineering to improve enzyme catalytic efficiency, this manuscript is interesting in the industrial aspect, as well. This manuscript is well-organized and specifically describes the active site structure. Therefore, this manuscript could be published in the current form. However, I suggest that the authors should address several minor issues to improve the quality of this manuscript.
<Minor points>
1. The author should clearly state which microorganism DszC used in this study originates from. In Abstract, Idenonella sakaiensis is mentioned. However, Rhodococcus erythropolis appears in Introduction.
2. The oligomeric state of DszC for enzyme working needs to be mentioned in an appropriate section. The authors showed the dimeric state in Figure 2. Is this the oligomeric sate in solution or just that in the crystallographic environment. If DszC exists as a dimer in solution, please mention it in the manuscript, along with the corresponding references.
3. Although many results including structural description are shown in this manuscript, they depend on computational simulations. Thus, the authors should mention the intrinsic limitations of these methodological tools, and the necessity to verify these results with other experimental methods in the future.
Author Response
In this manuscript, the authors identified potential binding sites for substrates of DszC related to crude oil biodessulfurization. The authors also propose the mechanism of feedback inhibition of DszC, based on the results of molecular docking and molecular dynamics simulations. Considering that these results can be applied to protein engineering to improve enzyme catalytic efficiency, this manuscript is interesting in the industrial aspect, as well. This manuscript is well-organized and specifically describes the active site structure. Therefore, this manuscript could be published in the current form. However, I suggest that the authors should address several minor issues to improve the quality of this manuscript.
- The author should clearly state which microorganism DszC used in this study originates from. In Abstract, Idenonella sakaiensis is mentioned. However, Rhodococcus erythropolis appears in Introduction.
Reply: The correct organism is Rhodococcus erythropolis. We have corrected the typo accordingly: “The Rhodococcus erythropolis (strain IGTS8) bacterium has a tremendous industrial interest as it can remove sulfur from crude oil through its four-enzyme (DszA-D) 4S metabolic pathway.”
- The oligomeric state of DszC for enzyme working needs to be mentioned in an appropriate section. The authors showed the dimeric state in Figure 2. Is this the oligomeric sate in solution or just that in the crystallographic environment. If DszC exists as a dimer in solution, please mention it in the manuscript, along with the corresponding references.
Reply: We now added the following sentence in the Introduction to clarify the point raised by the reviewer. In page 3, it can be read:
“DszC is functional as a homodimer,[37] although it has also been determined as a tetramer (dimer of homodimers) from gel filtration and X-ray experiments.[38,39]”
We also added additional structural information to briefly address the oligomerization of DszC. In page 3, it can now be read:
“The enzyme binds a flavin mononucletide (FMN) cofactor at the interface of the homodimer and the DBT substrate in a large pocket composed of an inner hydrophobic chamber and an outer hydrophilic chamber.[39] From the available structural data on the enzyme, the oligomerization of DszC occurs prevalently by a C-terminal domain encompassing residues Ala236-Ser417,[38,39] with more specific contributions from an Arg338 located on an α-helix close to the DBT binding pocket and the Tyr410-Ser417 terminal loop.[39] A conformational shift after FMN binding by the Gln280-Asp295 loop, contributing for FMN binding, and the Ser131-Lys142 loop of another monomer would then lead to a close conformation ready for catalysis.[38,40] Mutation of any of the key catalytic residues identified from QM/MM calculations by Barbosa et al. (Ser163, His92, Tyr96, His391, and Asn129) or residues contributing either for FMN binding or DszC dimerization, namely Arg338 or the Tyr410-Ser417 terminal loop led to loss of enzyme activity.[38]”
- Although many results including structural description are shown in this manuscript, they depend on computational simulations. Thus, the authors should mention the intrinsic limitations of these methodological tools, and the necessity to verify these results with other experimental methods in the future.
Reply: We now added a brief discussion at the beginning of the Results and Discussion section. In page 6, it can be read:
“We stress that our MD simulations are limited by the short simulation time when comparing to binding/unbinding events, which take microsseconds to seconds to occur, and do not take into account complex oligomerization events in solution, depending on the available concentration of DszC, or interactions with other molecules/structures in the bacterial cytosol. Such events would require either enhanced sampling simulation techniques,[46] only affordable in exascale computing platforms, or multiscale resolution methods,[47] which also compromise the molecular detail of the interactions taking place between the DszC and 2-HBP and HBPS. Hence, the results of our work should establish a groundwork for subsequent study with experimental methods, either in vitro or in vivo, that may provide further structural, kinetic or thermodynamic data to complement our findings.”
