Giardia intestinalis deoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

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   Review Commons

   Mar 27, 2024

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   Dear Editor,

   We have addressed the points and concerns raised by the reviewers and wish to thank them for their effort and time. We agree with all the comments and suggestions, which resulted in a significant improvement of the manuscript. Below, we provide a point-by-point response to all comments.

   Sincerely,

   Anders Hofer, corresponding author

   ---

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Dear Editor,

   We have addressed the points and concerns raised by the reviewers and wish to thank them for their effort and time. We agree with all the comments and suggestions, which resulted in a significant improvement of the manuscript. Below, we provide a point-by-point response to all comments.

   Sincerely,

   Anders Hofer, corresponding author

   ---

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). The authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this work will be paramount aid in better understanding of the reaction mechanism, especially in the context of molecules which can be used as inhibitors of such a crucial enzyme (metabolic vulnerability for this parasite).

   This manuscript, in its current form does not require additional experiments but I would like to have a few aspects corrected/clarified, before it can be accepted for publication:

   Line 30: "whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower." Better not to use term "affinity" when KM or kcat/KM are implied (unless ITC was used to measure true Kds).

   -This is a good point, and we are now using KM values in all instances were actual numbers are implied and only kept the word affinity in cases where it is discussed in more general terms.

   Line 31: "Deoxyadenosine analogues halogenated at the 2- and/or 2´-positions were also potent substrates, with comparable EC50 values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites." Not sure this sentence is clear as written.

   -We have now rewritten the sentence as follows: "Deoxyadenosine analogues halogenated at the 2- and/or 2´-positions were also potent substrates with comparable EC50 values on cultured G. intestinalis cells as metronidazole, the first line treatment today, with the additional advantage of being effective against metronidazole-resistant parasites."

   Line 55: "..G. intestinalis (synonymous to G. lamblia and G. duodenalis)..". Very nice that authors provide this information as it is usually a point of confusion i.e. multiple names for the same organism.

   -Thanks a lot, we are happy that you liked it.

   Line 61 and above as well: "Treatment regimes are mainly based on metronidazole and to a lesser extent other 5-nitroimidazoles...". MT is introduced a bit sporadically, and not completely clear which enzyme it inhibits and its mode of action." Common knowledge is that MT is known for its action in aerobic parasites/bacteria and known as Flagyl, where it is mode of action was linked to "activation" due to microaerophilic conditions. Maybe MT can be introduced after text starting from Line 71?

   -The description of metronidazole is adjusted as following: "Metronidazole (Flagyl) is the most commonly used drug to treat giardiasis and selectively kills the parasite and other anaerobic organisms by forming free radicals under oxygen-limited conditions, but it has side effects such as nausea, abdominal pain, diarrhea, and in some cases neurotoxicity reactions."

   Line 70: what is "cyst-wall"?

   -It is a cell wall consisting of three major cyst wall proteins and N-acetylgalactosamine. We have adjusted the sentence to the following to make the term clearer: The trophozoites can also secrete material to form a cyst wall and go through two rounds of DNA replication to form cysts, which contain four nuclei and a 16N genome per cell (4N in each nucleus).

   Line 90: "The reaction is catalyzed by deoxyribonucleoside kinases (dNKs), which are.." I really do not like when in order to find a reaction which is catalyzed by an enzyme in a particular study one needs to dive into the literature, sometimes it requires a lot of time as in most of recent papers on the subject reactions catalyzed are not listed. Please add a Figure or a panel with reactions catalyzed by both dNKs families.

   -It is a good idea and we have now added a figure (Fig. 1), which compares the deoxyribonucleotide metabolism of G. intestinalis with mammalian cells. The different deoxyribonucleoside kinases in the parasite and mammalian cells are included in the figure.

   Line 96: "..was found to have a ~10-fold higher affinity to thymidine.." as I mentioned above I really do not like the usage of "affinity", when actually low KM is implied.

   -It is corrected now (see above).

   Line 113: "This does not match the current knowledge that there are three dNKs in total whereof one completely specific for thymidine. The lack of knowledge about these essential enzymes in the parasite has hampered the understanding of Giardia deoxyribonucleoside metabolism and hence its exploitation as a target for antiparasitic drugs." Very good rationale, as I mentioned above, I think a Figure needs to be introduced that depicts different enzymes involved in deoxyribonucleoside metabolism (both TK1 and non- TK1 members) in Giardia with clearly labeled all known paralogs and corresponding enzymatic reactions.

   -Thanks a lot for the suggestion. Information about the different dNKs in G. intestinalis with mammalian cells for comparison is included in the new figure (Fig. 1).

   Line 132: Odd designation of supplementary figures, usually it is "Fig. S1" etc. The legend for Fig. S1 is not adequate, please add description of species and name of enzymes for all sequences shown. Also each sequences in alignment should start with number (a.a. number) as it is not clear if a full sequence is shown or not. Overall comment about the multiple sequence alignment (relevant to Fig. S1): with such a small number of sequences it is very hard to make any substantial predictions about conserved regions etc.

   -Thanks for the suggestions. We have now included more sequences, sequence numbering, and description of species as well as enzyme names. Some other changes are also that we have now used the same G. intestinalis dAK sequence in the alignment as in the experiments (same strain and accession number), and that we have made a realignment using Clustal W instead of Clustal Omega (gives better alignment of the termini). The designation of supplementary figures is according to the style of PLoS journals.

   Fig. 1 and elsewhere: I will prefer that all bar graphs show individual values + the error bar (if possible);

   -We have now added individual values to the bar graphs.

   I do not have any issues with X-ray data and cryo-EM studies (refinement statistics, particles classification etc).

   **Referees cross-commenting**

   I also agree with all the comments provided by Reviewer 2 and very pleased to see that we were very similar in our evaluations.

   Reviewer #1 (Significance (Required)):

   In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). The authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this work will be paramount aid in better understanding of the reaction mechanism, especially in the context of molecules which can be used as inhibitors of such a crucial enzyme (metabolic vulnerability for this parasite).

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   Summary:

   Ranjbarian et al. investigated a non-TK1-Like deoxyribonucleoside kinase (dNK) found in the protozoan parasite Giardia intestinalis. They used enzyme kinetic assays on heterologously expressed Gi dNK in E. coli to determine which deoxyribonucleotides were most likely physiological substrates for the enzyme. Their characterization revealed that this Gi dNK has a strong affinity to deoxyadenosine. They further investigated the affinity and activity of the dNK on deoxyadenosine analogues, some of which have known pharmaceutical utility. Finally, using a combination of crystallography, cryo-EM, chromatography, and mass photometry, they reveal that unlike other dNKs, Gi dNK forms a tetramer. They characterize important regions required for tetramerization and postulate that this tetramerization evolved to provide Gi dNK with a heightened affinity for deoxyadenosine.

   Major comments and questions:

   • The claims in this manuscript are well-supported, and I found no major issues with experimental methods. • The authors provide a structure of tetrameric dNK and suggest that this tetramer leads to the increased affinity to substrate compared to non-giardia dNKs. They also show through mutations that removing the novel dimerization regions decreases substrate affinity by 100-fold. However, I was left unclear about why the tetramer would lead to such high affinity for substrate compared to two dimers. This is especially notable, since the authors state that there are no signs of cooperativity, which is a common way that oligomerization may lead to heightened affinity. If the authors have no current evidence explaining this, they can consider adding a short amount of discussion speculating on the mechanism and future directions of study. -Thanks for this suggestion. We have now added a section in the last paragraph of the discussion where we speculate on the subject.

   Minor comments and questions:

   • The authors state that dATP acts as a mixed inhibitor and not a simple competitive inhibitor, and that previous studies have shown that this is because the dNTP competes in two locations (line 163). Is it also possible that competitive inhibition + allosteric regulation could be causing this behavior instead? -It is true that this can be theoretically explained in many ways. In fact, many allosteric regulators affect both the Vmax and Km values. However, in all studied dNKs, the dNTP acts as a dual competitor and no proper allosteric regulation with a separate allosteric site has ever been observed so far. We have rephrased this part as following to make it clear: "Mixed inhibition is often the result of allosteric regulation but studies of other dNKs have shown that this is not the case [17]. Instead, the far-end dNTP product gives a dual inhibition where the deoxyribonucleoside moiety competes with the substrate and the phosphate groups mimic those of ATP but coming from the opposite direction."

   • In the introduction (line 93), non-TK1-like dNKs are described as "not structurally related to TK1-like". This left me unclear, are they still interrelated among themselves? -We have added the following sentence for clarification: "The non-TK1-like dNKs are further subdivided into a monophyletic group of canonical non-TK1-like dNKs and a second group with thymidine kinases from Herpesviridae, which are structurally related to the canonical group but share very little amino acid sequence homology."

   • I was left confused by lines 106-116 in the introduction, where the specificities of dNKs in giardia are discussed. This is touched upon again in the discussion, but it was not clear here that there are several deoxyribonucleotides unaccounted for. -We think this should be clear now with the added Fig. 1 where the dNKs are shown.

   • When describing enzyme assays (Line 145), the authors say there is no salt dependence, but there looks to be MgCl2 always included in the assays (presumably for the ATP). -This is a good point and something we have overlooked when the sentence was written (Mg2+ is required). We have now corrected the sentence as follows: "Based on initial enzyme activity studies, it was confirmed that the assay did not have any specific requirements regarding K+, Na+, NH4+, acetate or reducing agents, and that it was linear with respect to time (S2 Fig)."

   • I was confused by the y-axis of Fig 2. How is enzyme activity lower when dAdo is added? I think I read "enzyme activity" as total substrate depleted, when it is actually referring exclusively to the given non-dAdo substrate in each column. -This is a very good point that we seem to have overlooked. We have now adjusted the y-axis title to "Indicated enzyme activity" and added the following sentence to the figure legend: "The recorded enzyme activities are for the substrates indicated on the x-axis (excluding the activity with the competing substrate)."

   • Lines 239 - 255 and Figure 3 were a little unclear to me. Specifically, I was having trouble following in the text which dimer is in the ASU, which is symmetry related, and matching those terms with which are canonical and non-canonical. -We agree with the reviewer and thank them for their comment. In order to improve the presentation of these results, we chose to extensively rearrange the figures and accompanying text. We now present the initial X-ray data together with the cryo-EM data in a new Figure 4 that focuses on the overall architecture of the tetramer. We realize that some of the nomenclature previously used in that figure was, as the reviewer pointed out, confusing and superfluous, and we have now simplified and unified it. The structural details of how the extended N- and C-termini interact with the neighboring subunit have been moved to the new figure 6 in order to present them just before the functional analyses of the consequences of truncating the termini. As a consequence of these changes to the figure layout, we made substantial changes in the organization of the text surrounding these figures, which also led to a clearer presentation. Since the changes to the figures and text are quite substantial, we would like to point out that they are only changes to the presentation, not to the data shown.

   • The authors suggest that in the experiment shown in Figure S9 (Line 285), low activity may be caused by minor impurities. I'm not sure why impurities would lower activity significantly. Could there be other differences in experimental conditions that are at play instead? -The sentence refers to a side activity (dATP dephosphorylation) which is not the normal reaction of deoxyadenosine kinase. We have rephrased the sentence to make it clearer: "The dATP-dephosphorylating activity was several orders of magnitude lower than the regular dAK activity (to phosphorylate deoxyadenosine) and was possibly catalyzed by other enzymes present as minor impurities in the protein preparation."

   • (Optional) From looking at the crystallography stats, I think the authors can potentially push the resolution more. At higher resolutions, Rmerge may become high, but depending on the data collection strategy, Eiger detectors can lead to high Rmerge just out of sheer data redundancy. Cc 1/2 can be a more useful metric in these contexts. -This is a good point and well spotted by the reviewer. Indeed, a CC1/2 of 0.802 suggests that the resolution can be pushed further. However, due to contaminating spots at higher resolutions the statistics significantly worsen when trying to push the resolution beyond 2.1 A, which is why we did not process the data to a higher resolution.

   • For Figure S8, the Polder map feature in Phenix is another option for showing ligand occupancy in an unbiased way. Did the authors try this? -We want to thank the reviewer for suggesting this. We have calculated a polder map using the Polder map feature in Phenix and both the resulting map and correlation coefficients support the presence of a dADP in the active site of monomer I. We added a section to the relative paragraph to include these new findings: "To increase our confidence that dADP was correctly placed within active site I, we calculated a polder map for dADP to test whether the b-phosphate density is correctly attributed or if it rather belongs to the bulk solvent. The resulting polder map and statistics support the placement of dADP in active site I with correlation coefficients of CC1,2=0.7627, CC1,3=0.9424, and CC2,3=0.7423 suggesting that the density does belong to dADP as CC1,3 > CC1,2 and CC1,3 > CC2,3 (S8 Fig.)."

   • It's disappointing that the tetramers show so much preferred orientation in the cryo-EM. With that said, while the nominal resolution is 4.8 Å, I think that with the streakiness the EM structure looks to have worse resolution than that. -We agree that the streakiness of the map is substantial. This is simply a result of the severe anisotropy of the map, which means that the resolution is probably worse than 4.8 Å in the "bad directions" of the map. The supplementary material (S9 Fig) clearly shows the preferred orientations leading to this problem. In the course of this study, we tried several methods to lessen the preferred orientation problem such as using graphene oxide-coated grids and collecting tilted data. However, when we got the crystal structure we saw no point in continuing these efforts. To address the comment of the reviewer, we extended the description of the EM map in the main text to say:

   "Due to strong preferred orientations, it was not possible to get an isotropic, high-resolution 3D structure of dAK using cryo-EM. The resulting 3D map had a nominal resolution of 4.8 Å, but a clearly anisotropic appearance probably reflecting lower resolution in the poorly resolved direction (S9 Fig)."

   **Referees cross-commenting**

   Overall, I agree with Reviewer #1's evaluation, and don't have any further suggestions or thoughts at this time.

   Reviewer #2 (Significance (Required)):

   Medical relevance: G. intestinalis is a parasite that causes 190 million cases of giardiasis per year. While treatable, there is evidence that giardia are developing a resistance to the main treatment at the moment, metronidazole. Thus, the authors provide a compelling case for the medical relevance of their investigation of Gi dNK for further pharmaceutical development. They provide further evidence for this by showing that several deoxyadenosine analogs bind the dNK and inhibit giardia growth. This work represents a very useful first step into a potential avenue for medical development. It's important to note that clinical studies are not within the purview of this research. However, in the discussion, the authors provide several comments on the promise of this avenue for future research.

   Conceptual, technical, and mechanistic relevance: Through biochemical and structural study, the authors provide a compelling framework to understand an enzyme that is very important to the unique lifestyle of giardia parasites. From an evolutionary standpoint, the authors provide insight into how giardia can survive even without major components of de novo DNA synthesis. The authors principally use well-established tools and techniques of the enzymology field. but do so to characterize a unique and previously uncharacterized enzyme system. This enzyme proves to be notable not just for its medical significance, but because it is unique among its family (non-TK1-like deoxynucleotide kinases) in its strong affinity for substrate and tetrameric quaternary structure. One relatively novel technique used in the study is mass photometry, which is a relatively new and exciting way to characterize native proteins at very low concentrations. Using this technique helps the authors overcome a common criticism of structural studies in which the high concentrations or crowding conditions of techniques like crystallography and cryo-EM may be inducing non-physiological oligomers.

   In summary, this work represents a meaningful addition to the protein structure-function literature. While it will principally be of interest to basic/fundamental researchers who study the mechanistic detail of protein function and evolution, it also provides a foundation for future translational work and antiparasitic drug design.

   Reviewer's background: I received my PhD in chemistry studying the structure and function of another enzyme key to DNA metabolism (except in giardia), ribonucleotide reductase. My background is in structural biology and biochemistry. I do not have sufficient expertise to comment on studies performed on G. intestinalis growth and susceptibility to deoxyadenosine analogs.

   Read the original source
2. 

   Review Commons

   Mar 27, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   ---

   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   Ranjbarian et al. investigated a non-TK1-Like deoxyribonucleoside kinase (dNK) found in the protozoan parasite Giardia intestinalis. They used enzyme kinetic assays on heterologously ex-pressed Gi dNK in E. coli to determine which deoxyribonucleotides were most likely physiological substrates for the enzyme. Their characterization revealed that this Gi dNK has a strong affinity to deoxyadenosine. They further investigated the affinity and activity of the dNK on deoxyadenosine analogues, some of which have known pharmaceutical utility. Finally, using a combination of crys-tallography, cryo-EM, chromatography, and mass …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   Ranjbarian et al. investigated a non-TK1-Like deoxyribonucleoside kinase (dNK) found in the protozoan parasite Giardia intestinalis. They used enzyme kinetic assays on heterologously ex-pressed Gi dNK in E. coli to determine which deoxyribonucleotides were most likely physiological substrates for the enzyme. Their characterization revealed that this Gi dNK has a strong affinity to deoxyadenosine. They further investigated the affinity and activity of the dNK on deoxyadenosine analogues, some of which have known pharmaceutical utility. Finally, using a combination of crys-tallography, cryo-EM, chromatography, and mass photometry, they reveal that unlike other dNKs, Gi dNK forms a tetramer. They characterize important regions required for tetramerization and pos-tulate that this tetramerization evolved to provide Gi dNK with a heightened affinity for deoxy-adenosine.

   Major comments and questions:

   • The claims in this manuscript are well-supported, and I found no major issues with experi-mental methods.
   • The authors provide a structure of tetrameric dNK and suggest that this tetramer leads to the increased affinity to substrate compared to non-giardia dNKs. They also show through mu-tations that removing the novel dimerization regions decreases substrate affinity by 100-fold. However, I was left unclear about why the tetramer would lead to such high affinity for substrate compared to two dimers. This is especially notable, since the authors state that there are no signs of cooperativity, which is a common way that oligomerization may lead to heightened affinity. If the authors have no current evidence explaining this, they can con-sider adding a short amount of discussion speculating on the mechanism and future direc-tions of study.

   Minor comments and questions:

   • The authors state that dATP acts as a mixed inhibitor and not a simple competitive inhibitor, and that previous studies have shown that this is because the dNTP competes in two loca-tions (line 163). Is it also possible that competitive inhibition + allosteric regulation could be causing this behavior instead?
   • In the introduction (line 93), non-TK1-like dNKs are described as "not structurally related to TK1-like". This left me unclear, are they still interrelated among themselves?
   • I was left confused by lines 106-116 in the introduction, where the specificities of dNKs in giardia are discussed. This is touched upon again in the discussion, but it was not clear here that there are several deoxyribonucleotides unaccounted for.
   • When describing enzyme assays (Line 145), the authors say there is no salt dependence, but there looks to be MgCl2 always included in the assays (presumably for the ATP).
   • I was confused by the y-axis of Fig 2. How is enzyme activity lower when dAdo is added? I think I read "enzyme activity" as total substrate depleted, when it is actually referring ex-clusively to the given non-dAdo substrate in each column.
   • Lines 239 - 255 and Figure 3 were a little unclear to me. Specifically, I was having trouble following in the text which dimer is in the ASU, which is symmetry related, and matching those terms with which are canonical and non-canonical.
   • The authors suggest that in the experiment shown in Figure S9 (Line 285), low activity may be caused by minor impurities. I'm not sure why impurities would lower activity sig-nificantly. Could there be other differences in experimental conditions that are at play in-stead?
   • (Optional) From looking at the crystallography stats, I think the authors can potentially push the resolution more. At higher resolutions, Rmerge may become high, but depending on the data collection strategy, Eiger detectors can lead to high Rmerge just out of sheer data redundancy. Cc 1/2 can be a more useful metric in these contexts.
   • For Figure S8, the Polder map feature in Phenix are another option for showing ligand oc-cupancy in an unbiased way. Did the authors try this?
   • It's disappointing that the tetramers show so much preferred orientation in the cryo-EM. With that said, while the nominal resolution is 4.8 Å, I think that with the streakiness the EM structure looks worse resolution than that.

   Referees cross-commenting

   Overall, I agree with Reviewer #1's evaluation, and don't have any further suggestions or thoughts at this time.

   Significance

   Medical relevance: G. intestinalis is a parasite that causes 190 million cases of giardiasis per year. While treatable, there is evidence that giardia are developing a resistance to the main treatment at the moment, metronidazole. Thus, the authors provide a compelling case for the medical relevance of their investigation of Gi dNK for further pharmaceutical development. They provide further evi-dence for this by showing that several deoxyadenosine analogs bind the dNK and inhibit giardia growth. This work represents a very useful first step into a potential avenue for medical develop-ment. It's important to note that clinical studies are not within the purview of this research. Howev-er, in the discussion, the authors provide several comments on the promise of this avenue for future research.

   Conceptual, technical, and mechanistic relevance: Through biochemical and structural study, the authors provide a compelling framework to understand an enzyme that is very important to the unique lifestyle of giardia parasites. From an evolutionary standpoint, the authors provide insight into how giardia can survive even without major components of de novo DNA synthesis.

   The authors principally use well-established tools and techniques of the enzymology field. but do so to characterize a unique and previously uncharacterized enzyme system. This enzyme proves to be notable not just for its medical significance, but because it is unique among its family (non-TK1-like deoxynucleotide kinases) in its strong affinity for substrate and tetrameric quater-nary structure. One relatively novel technique used in the study is mass photometry, which is a relatively new and exciting way to characterize native proteins at very low concentrations. Using this technique helps the authors overcome a common criticism of structural studies in which the high concentrations or crowding conditions of techniques like crystallography and cryo-EM may be inducing non-physiological oligomers.

   In summary, this work represents a meaningful addition to the protein structure-function literature. While it will principally be of interest to basic/fundamental researchers who study the mechanistic detail of protein function and evolution, it also provides a foundation for future transla-tional work and antiparasitic drug design.

   Reviewer's background: I received my PhD in chemistry studying the structure and function of another enzyme key to DNA metabolism (except in giardia), ribonucleotide reductase. My back-ground is in structural biology and biochemistry. I do not have sufficient expertise to comment on studies performed on G. intestinalis growth and susceptibility to deoxyadenosine analogs.

   Read the original source
3. 

   Review Commons

   Mar 27, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). Authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). Authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this work will be paramount aid in better understanding of reaction mechanism, especially in the context of molecules which can be used as inhibitors of such crucial enzyme (metabolic vulnerability for this parasite).

   This manuscript, in its current form does not require additional experiments but I would like to have a few aspects corrected/clarified, before it can be accepted for publication:

   Line 30: "whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower." Better not to use term "affinity" when KM or kcat/KM are implied (unless ITC was used to measure true Kds).

   Line 31: "Deoxyadenosine analogues halogenated at the 2- and/or 2´-positions were also potent substrates, with comparable EC50 values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites." Not sure this sentence is clear as written.

   Line 55: "..G. intestinalis (synonymous to G. lamblia and G. duodenalis)..". Very nice that authors provide this information as it is usually a point of confusion i.e. multiple names for the same organism.

   Line 61 and above as well: "Treatment regimes are mainly based on metronidazole and to a lesser extent other 5-nitroimidazoles...". MT is introduced a bit sporadically, and not completely clear which enzyme it inhibits and its mode of action." Common knowledge is that MT is known for its action in aerobic parasites/bacteria and known as Flagyl, where it is mode of action was linked to "activation" due to microaerophilic conditions. Mayve MT can be introduced after text starting from Line 71?

   Line 70: what is "cyst-wall"?

   Line 90: "The reaction is catalyzed by deoxyribonucleoside kinases (dNKs), which are.." I really do not like when in order to find a reaction which is catalyzed by an enzyme in a particular study one needs to dive into the literature, sometimes it requires a lot of time as in most of recent papers on the subject reactions catalyzed are not listed. Please add a Figure or a panel with reactions catalyzed by both dNKs families.

   Line 96: "..was found to have a ~10-fold higher affinity to thymidine.." as I mentioned above I really do not like the usage of "affinity", when actually low KM is implied.

   Line 113: "This does not match the current knowledge that there are three dNKs in total whereof one completely specific for thymidine. The lack of knowledge about these essential enzymes in the parasite has hampered the understanding of Giardia deoxyribonucleoside metabolism and hence its exploitation as a target for antiparasitic drugs." Very good rationale, as I mentioned above, I think a Figure needs to be introduced that depicts different enzymes involved in deoxyribonucleoside metabolism (both TK1 and non- TK1 members) in Giardia with clearly labeled all known paralogs and corresponding enzymatic reactions.

   Line 132: Odd designation of supplementary figures, usually it is "Fig. S1" etc. The legend for Fig. S1 is not adequate, please add description of species and name of enzymes for all sequences shown. Also each sequences in alignment should start with number (a.a. number) as it is not clear if a full sequence is shown or not. Overall comment about the multiple sequence alignment (relevant to Fig. S1): with such a small number of sequences it is very hard to make any substantial predictions about conserved regions etc.

   Fig. 1 and elsewhere: I will prefer that all bar graphs show individual values + the error bar (if possible);

   I do not have any issues with X-ray data and cryo-EM studies (refinement statistics, particles classification etc).

   Referees cross-commenting

   I also agree with all the comments provided by Reviewer 2 and very pleased to see that we were very similar in our evaluations.

   Significance

   In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). Authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this work will be paramount aid in better understanding of reaction mechanism, especially in the context of molecules which can be used as inhibitors of such crucial enzyme (metabolic vulnerability for this parasite).

   Read the original source
4. 

   Version published to 10.1101/2023.12.18.572228v1 on bioRxiv

   Dec 18, 2023