Is dUTPase Enzymatic Activity Truly Essential for Viability?

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. As a review article, giving some limitations or research directions in the future would be helpful. 

2. The different types of dUTPase were compared. I suggest to show the results in a figure or table to help readers understand them more directly. 

3. The organization of the manuscript could be improved. For example, some paragraphs only have one sentences. 

4. "Caused their wide prevalence in pathogen organisms, understanding the structural 66
and functional characteristics of dUTPases provides valuable insights for their potential 67
use as new targets in antimicrobial, antiviral and possibly anticancer therapies [24-28]." should be "Considering their wide prevalence in pathogen organisms, understanding the structural 66
and functional characteristics of dUTPases provides valuable insights for their potential 67
use as new targets in antimicrobial, antiviral and possibly anticancer therapies [24-28]."

Author Response

1. As a review article, giving some limitations or research directions in the future would be helpful. 

Response1:  For now this is just our hypothesis and we will work on testing it.

2. The different types of dUTPase were compared. I suggest to show the results in a figure or table to help readers understand them more directly. 

Response2: It is not entirely clear by what other parameters to compare? In our opinion, Figures 2 and 3 provide the maximum comparison of all the presented enzyme groups.

3. The organization of the manuscript could be improved. For example, some paragraphs only have one sentences. 

"Caused their wide prevalence in pathogen organisms, understanding the structural and functional characteristics of dUTPases provides valuable insights for their potential use as new targets in antimicrobial, antiviral and possibly anticancer therapies [24-28]." should be "Considering their wide prevalence in pathogen organisms, understanding the structural and functional characteristics of dUTPases provides valuable insights for their potential use as new targets in antimicrobial, antiviral and possibly anticancer therapies [24-28]."

Response3: The recommendations have been taken into account and included in the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to the authors:

1. Abstract Lines 16-17 repetition of word providing should be corrected
2. Line 33, “Members of this family are found in all living organisms, as well as in a number of eukaryotic viruses and bacteriophages” this is not true as it is correctly presented in Line 320

“At the same time, bioinformatic analysis of bacterial and archaeal genomes has revealed amazing fact that the absence of the dut gene is not an exceptionally rare phenomenon among the genomes analyzed [6].” thus the sentence in Line 33 should be corrected.

3. Lines 40-41 uracil in DNA could change the binding of transcription factors, thus it is not indistinguishable from norma DNA. Here it should be mentioned that uracil is excised because it is the product of cytosine deamination which is a mutagenic process.
4. Line 107 correct display of the mechanism is mechanism is S_N2
5. The difference in the reaction mechanism of the trimer and dUTPases should be displayed on Fig 1 similarly to that in Nagy et al 2014 FEBS J (PMID: 25052017).
6. Detailed description of enzymatic mechanism of dimer dUTPases according to Hemsworth et al Biochem J. 2013 (PMID: 24001052) and other should be provided.
7. Line 131 correctly: Homo sapiens
8. Lines 153-162 the first discovery of the role of metal ion in dUTPase catalysis by Mustafi et al PNAS 2003 (PMID 12721364) should be cited.
9. Line 162. “in their apo-form or in complex with dUMP, do not contain magnesium ions [39, 42]”, this is not true entirely, these structures does not contain magnesium in their active site. But apo structures under PDB IDs: 2XCD,1DUT and 4OOQ contein magnesium ion in their central channel. (cf also Leveles et al Acta Crys D 2013 PMID: 24311572)
10. Description of covalent homotrimer of Drosophila virilis is missing cf (Benedek et al FEBS J 2016 27380921)
11. On Figure 2 Type 2 trimer dUTPases in which 2 subunits form the active site should be represented
12. Lines 233-238. This mutant is does not raise any particular interest, it is quite obvious that if one exchange a small residue in the active site to a bulky one that could hinder substrate binding. If this example is shown, other similar studes from the trimer dUTPase field should be included like: Harris et al JMB 1999 PMID: 10329142, Szabó et al Sci rep 2016 PMID: 27063406 (human DUT A98F), Hirmondo et al Sci rep 2017 PMID 28729658 (A115F mutant of M. tuberculosis dcd-dut) among perhaps others.
13. Lines 280-290. The structure of PRV dUTPase should be shown, as it is difficult to follow the text without that. This is indeed an interesting dimer of 2 dUTPase chains resembling monomer DUTs. The authors of Ref 33 display six homologous motifs within the herpes virus family, but those does not match with the general consensus of literature presented for example in ref 42 (motif 3 of PRV is not near to the active site, in other cases conserved motifs forming the active site are listed also in this manuscript). In light of this, the sentence in Lines 283-285 should be omitted, or only the similarity PRV dut to EBV dut active site forming motifs should be emphasized.
14. Lines 375-377 “. At the same time, mutant forms of dUTPases lacking motif VI retain enzymatic activity but lose the ability to induce SaPI mobilization.” This is factually incorrect. Maiques et al NAR 2016 PMID: 27112567 write “To check the role of motif VI in Dutϕ11, we replaced residues 96–123 with Ser-Asn and produced the mutant Dutϕ11ΔVI (Figure 1). This mutant displays superior enzymatic activity to that of the wild-type version but a slightly reduced capacity to induce SaPIbov1 (Table 2 and Figure 2B)”. So insert-less Dutϕ11 is capable of SaPI induction. Also interestingly in Ref46 during phage evolution under SaPI interference pressure only mutations in the core region (not in the insert) like I75N, G164S, A32T of the enzyme evolve to avoid SaPI interference, which also argues against the insert in SaPI induction.
15. Lines 411-412 “Conversely, there are found proteins with dUTP-binding sites that do not catalyze dUTP hydrolysis.” References and some more details are needed here.
16. Lines 425-427 “Moreover, given that the absence of dUTP hydrolysis itself does not cause critical harm to the organism, unlike dut gene knockout, it is plausible that the key factor lies in the ability to bind dUTP.” i) Moonlighting functions via protein-protein binding are also plausible ii) when dUTP is bound to dUTPase it is immediately hydrolyzed in a relatively fast chemical step so it is highly unlikely that “the key factor lies in the ability to bind dUTP”. cf for example Tóth et al J Biol Chem 2007, PMID: 17848562.
17. Lines 432-446 to be omitted. dUTPase is a dUTP sanitizing enzyme thus it hydrolyze dUTP relatively fast and without protein cofactors as compared to GTPases (hDUT k_cat is 6.4 s^-1, while human p21-RAS k_cat 0.0055 s^-1). Both the dUTP and dUMP bound state is transient (dUMP has low affinity to dUTPases), compared to GTPases which are GTP or GDP bound most of the time, due to the higher affinity and lower reactivity. For more details on this cf also Szabó et al NAR 2014 PMID: 25274731, especially Page 7, Fig 3 and Table S3.
18. In the abstract kinetic parameters are mentioned but none is displayed in the manuscript, either complement the manuscript or refine the abstract.

 

Author Response

Abstract Lines 16-17 repetition of word providing should be corrected

Response1: Corrected

 

Line 33, “Members of this family are found in all living organisms, as well as in a number of eukaryotic viruses and bacteriophages” this is not true as it is correctly presented in Line 320

“At the same time, bioinformatic analysis of bacterial and archaeal genomes has revealed amazing fact that the absence of the dut gene is not an exceptionally rare phenomenon among the genomes analyzed [6].” thus the sentence in Line 33 should be corrected.

Response2 : «Members of this family are found in all living organisms, as well as in a number of eukaryotic viruses and bacteriophages [1-5]» changed to «dUTPases are ubiquitous across all domains of life, as well as in numerous eukaryotic viruses and bacteriophages [1-5]»

 

Lines 40-41 uracil in DNA could change the binding of transcription factors, thus it is not indistinguishable from norma DNA. Here it should be mentioned that uracil is excised because it is the product of cytosine deamination which is a mutagenic process.

Response3: Corrected

 

Line 107 correct display of the mechanism is mechanism is S_N2

Response4: Corrected

 

The difference in the reaction mechanism of the trimer and dUTPases should be displayed on Fig 1 similarly to that in Nagy et al 2014 FEBS J (PMID: 25052017).

Detailed description of enzymatic mechanism of dimer dUTPases according to Hemsworth et al Biochem J. 2013 (PMID: 24001052) and other should be provided.

Response5: We prepare additional panels Fig2e and Fig 3c.

 

Line 131 correctly: Homo sapiens

Response 6: Corrected

 

Lines 153-162 the first discovery of the role of metal ion in dUTPase catalysis by Mustafi et al PNAS 2003 (PMID 12721364) should be cited.

Response 7: The recommendations have been taken into account and included in the manuscript

 

Line 162. “in their apo-form or in complex with dUMP, do not contain magnesium ions [39, 42]”, this is not true entirely, these structures does not contain magnesium in their active site. But apo structures under PDB IDs: 2XCD,1DUT and 4OOQ contein magnesium ion in their central channel. (cf also Leveles et al Acta Crys D 2013 PMID: 24311572)

Response8:  “in their apo-form or in complex with dUMP, do not contain magnesium ions [39, 42]” changed to “in their apo-form or in complex with dUMP, do not contain magnesium ions in their active site [39, 42]”

 

Description of covalent homotrimer of Drosophila virilis is missing cf (Benedek et al FEBS J 2016 27380921)

Response 9: The recommendations have been taken into account and included in the manuscript

 

On Figure 2 Type 2 trimer dUTPases in which 2 subunits form the active site should be represented

Response10:

This is difficult to show in one picture and therefore it was described in the text.

“In most cases, motif V originates from the third subunit, which is not adjacent to the active site. However, exceptions have been reported (e.g., African swine fever virus dUTPase, white spot syndrome virus (wDUT), and Plasmodium falciparum dUTPase), where the C-terminal region of the protein exhibits a different fold [39-41]. In these cases, motif V contributes to the active site architecture in conjunction with motif III from the same subunit. This unique architectural organization, wherein the formation of the active site depends directly on proper trimer assembly, is unusual among enzymes and underscores the structural and functional sophistication of trimeric dUTPases”

 

Lines 233-238. This mutant is does not raise any particular interest, it is quite obvious that if one exchange a small residue in the active site to a bulky one that could hinder substrate binding. If this example is shown, other similar studes from the trimer dUTPase field should be included like: Harris et al JMB 1999 PMID: 10329142, Szabó et al Sci rep 2016 PMID: 27063406 (human DUT A98F), Hirmondo et al Sci rep 2017 PMID 28729658 (A115F mutant of M. tuberculosis dcd-dut) among perhaps others.

Response 11: paragraph removed

 

Lines 280-290. The structure of PRV dUTPase should be shown, as it is difficult to follow the text without that. This is indeed an interesting dimer of 2 dUTPase chains resembling monomer DUTs. The authors of Ref 33 display six homologous motifs within the herpes virus family, but those does not match with the general consensus of literature presented for example in ref 42 (motif 3 of PRV is not near to the active site, in other cases conserved motifs forming the active site are listed also in this manuscript). In light of this, the sentence in Lines 283-285 should be omitted, or only the similarity PRV dut to EBV dut active site forming motifs should be emphasized.

Response 12: The quality and reliability of this structure (pdb id 8ZWQ) raises serious questions. R-Value Work: 0.277 R-Value Free: 0.339 at resolution 2.23 Å this is very strange. In addition, electron density analysis showed that the structure is very poorly fitted. The position of the ligand is also highly questionable. That is why we mentioned but did not focus on this structure.

A notable inconsistency exists in the nomenclature and sequential order of motifs within the polypeptide chain of monomeric dUTPases. As clearly demonstrated in Ref. 42, the conserved motif III—a hallmark of homotrimeric dUTPases—is shifted towards the N-terminus in the monomeric enzymes, resulting in a distinct motif order: III, I, II, IV, V.

This established nomenclature, however, was redefined in Ref. 33. The authors systematically renamed the motifs as follows: motif III to motif 1, motif I to motif 2, motif II to motif 4, motif IV to motif 5, and motif V to motif 6. This renumbering creates potential for confusion. For instance, in Figure 3C of Ref. 33, the motifs labeled 1, 2, 4, and 5 correspond directly to the previously known motifs III, I, II, and IV, respectively. Furthermore, in Figure 3A the authors introduced an additional element, designated as motif 3, which is noted to be conserved among monomeric dUTPases but does not contribute to the active site architecture.

 

Lines 375-377 “. At the same time, mutant forms of dUTPases lacking motif VI retain enzymatic activity but lose the ability to induce SaPI mobilization.” This is factually incorrect. Maiques et al NAR 2016 PMID: 27112567 write “To check the role of motif VI in Dutϕ11, we replaced residues 96–123 with Ser-Asn and produced the mutant Dutϕ11ΔVI (Figure 1). This mutant displays superior enzymatic activity to that of the wild-type version but a slightly reduced capacity to induce SaPIbov1 (Table 2 and Figure 2B)”. So insert-less Dutϕ11 is capable of SaPI induction. Also interestingly in Ref46 during phage evolution under SaPI interference pressure only mutations in the core region (not in the insert) like I75N, G164S, A32T of the enzyme evolve to avoid SaPI interference, which also argues against the insert in SaPI induction.

Response 13: The noted clarifications concerning the role of additional motif VI have been included in the manuscript.

The amino acid substitutions reported in 80α Dut indeed lead to a decrease in the efficiency of SaPI mobilization, despite the fact that they do not fall within motif VI. Since the authors of Ref. 46 note that these substitutions also reduce enzymatic activity, it can be proposed that they may affect the folding of the polypeptide chain. This altered folding could, in turn, lead to the observed decrease in the protein's ability to induce SaPI mobilization.

 

Lines 411-412 “Conversely, there are found proteins with dUTP-binding sites that do not catalyze dUTP hydrolysis.” References and some more details are needed here.

Response14: phrase removed

 

Lines 425-427 “Moreover, given that the absence of dUTP hydrolysis itself does not cause critical harm to the organism, unlike dut gene knockout, it is plausible that the key factor lies in the ability to bind dUTP.” i) Moonlighting functions via protein-protein binding are also plausible ii) when dUTP is bound to dUTPase it is immediately hydrolyzed in a relatively fast chemical step so it is highly unlikely that “the key factor lies in the ability to bind dUTP”. cf for example Tóth et al J Biol Chem 2007, PMID: 17848562.

Response15:  changed to “Moreover, given that the absence of dUTP hydrolysis itself does not cause critical harm to the organism, unlike the knockout of the dut gene, it is likely that the presence of the enzyme itself is crucial, and even the ability to bind dUTP may be sufficient”

 

Lines 432-446 to be omitted. dUTPase is a dUTP sanitizing enzyme thus it hydrolyze dUTP relatively fast and without protein cofactors as compared to GTPases (hDUT kcat is 6.4 s-1, while human p21-RAS kcat 0.0055 s-1). Both the dUTP and dUMP bound state is transient (dUMP has low affinity to dUTPases), compared to GTPases which are GTP or GDP bound most of the time, due to the higher affinity and lower reactivity. For more details on this cf also Szabó et al NAR 2014 PMID: 25274731, especially Page 7, Fig 3 and Table S3.

Response16: changed to:

In light of this concept, the perception of dUTP as a substrate requiring processing undergoes a shift. From being merely a nucleotide — a building block of DNA or an auxiliary supplier of dTTP precursors — it transitions to the status of a molecule used for additional alternative purposes. A comparable example can be found in the roles of GTP and ATP nucleotides. The classes of GTPases and ATPases encompass a wide variety of proteins that utilize the energy derived from GTP or ATP hydrolysis to perform their primary functions (here we do not take into account the G-proteins but keep in mind such as transcription and translation factors and enzymes). Structurally, these proteins share domains for binding their respective nucleotides, but their primary functions can differ significantly. In this context, the dUTP molecule may also be meant as an effector who’s binding and/or hydrolysis is essential for executing another protein function. Here, the "signal transmission" could be mediated by structural rearrangements upon dUTP binding, such as increased mobility in the C-terminal region of trimeric dUTPases or the transition of two-domain enzymes from an open to a closed form

In the abstract kinetic parameters are mentioned but none is displayed in the manuscript, either complement the manuscript or refine the abstract.

Response17: changed to

 In this review, we have tried to touch upon only a few points from the classical description of enzymes of the dUTPase family and have added some additional functional properties of a number of representatives of this family.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have solved all my concerns. 

Author Response

We are reciprocally grateful to the reviewer for their scrupulous reading of our manuscript

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

I am grateful for the efforts of the authors of the mansucript entitled Is dUTPase Enzymatic Activity Truly Essential for Viability? made to significantly enhance their manuscript. The additional mechanistic data and modification of the text would be very useful for the readers of this work.

Now I have only a few minor issues to be addressed before publication.

In their reply the authors declared that the structure of 8ZWQ is raises serious questions, I agree and strongly suggest the authors to comment/refer on this in their manuscript.

Minor corrections:

Line 16 “subsequential classification” → subsequent classification

Line 19 “the fact of existence of such extra functions” → the existence of such extra functions

Line 93 “Systematization of information regarding of dUTPase structure” → Systematization of information regarding dUTPase structure

Line 98 “high structural diversity inside enzyme family” → should be within the enzyme family

Line 356 “From all the known data it is deserve special segregating…” → From all the known data it deserves special emphasis that…

Line 438 “mutants unable to hydrolyze dUTP are frequently survival” → mutants unable to hydrolyze dUTP are frequently viable

Line 479 “dUTP primarily acts as a effector” → dUTP primarily acts as an effector

Line 474 “who’s binding and/or hydrolysis” → whose binding and/or hydrolysis

 

Author Response

In their reply the authors declared that the structure of 8ZWQ is raises serious questions, I agree and strongly suggest the authors to comment/refer on this in their manuscript.

Answer:

Regarding the structure pdb id 8ZWQ, we couldn't avoid describing it in our manuscript since the structure was published and its analysis has already been cited by various authors. But if to comment its quality, we would have needed to refine it and analyze the changes. This work is beyond the scope of our review and should be presented in a separate article with another subject.

 

Minor corrections:

Line 16 “subsequential classification” → subsequent classification

Answer: Corrected

Line 19 “the fact of existence of such extra functions” → the existence of such extra functions

Answer: Corrected

Line 93 “Systematization of information regarding of dUTPase structure” → Systematization of information regarding dUTPase structure

Answer: Corrected

Line 98 “high structural diversity inside enzyme family” → should be within the enzyme family

Answer: Corrected

Line 356 “From all the known data it is deserve special segregating…” → From all the known data it deserves special emphasis that…

Answer: Corrected

Line 438 “mutants unable to hydrolyze dUTP are frequently survival” → mutants unable to hydrolyze dUTP are frequently viable

Answer: Corrected

Line 479 “dUTP primarily acts as a effector” → dUTP primarily acts as an effector

Answer: Corrected

Line 474 “who’s binding and/or hydrolysis” → whose binding and/or hydrolysis

Answer: Corrected

 

 

We are reciprocally grateful to the reviewer for their scrupulous reading of our manuscript and their rigorous analysis of its content, presentation and even linguistics. Thanks to this comprehensive work, our manuscript has undoubtedly been significantly improved and refined.