PONE-D-22-24612New Structure of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis suggest a novel catalytic mechanism for the entire classPLOS ONE

Dear Dr. Abad-Zapatero,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper entitled “New Structure of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis

suggest a novel catalytic mechanism for the entire class” presents structures of FBPase II

from Francisella tularensis resolved using the crystallographic technique. According to the authors, the structures represent the active state of the enzyme, containing metal cofactor Mn2+ and complexed with the product of the reaction fructose-6-phosphate (F6P), and the substrate fructose-1,6-bisphosphate (F1,6P2). Their analysis of the structures presented in the manuscript suggests a novel mechanism of catalysis for FBPases II.

The manuscript is clearly written and demonstrates a convincing set of data supporting the conclusions. The object of study (FBPase II from Francisella tularensis) is not only interesting for biochemists/biologists but it may be also important from medical and public safety points of view.

While I have no doubts that the manuscript is acceptable for publication, I have some questions concerning conditions of the crystallization. The authors speculate about the mechanism of the reaction based on structures achieved by crystallization in the presence of very high concentrations of the product and the substrate of the reaction. However, it has been shown that high titer of the substrate can “inhibit” the rate of the reaction. It has been hypothesized that F1,6P2 at high concentrations binds to mammalian FBPases in slightly different manner than it does at low concentrations and this results in a lower kcat (“b x kcat, where b<1). I do not know if such “inhibition by substrate” has ever been observed in the case of FBPase II, but if it has, the structures resolved by the authors represent the enzyme which catalytic site is occupied by the substrate in a configuration suboptimal for catalysis.

Obviously, it is still very valuable structure but the mechanism of catalysis may be slightly different.

On the other hand, Francisella tularensis is an intracellular pathogen and the titer of F1,6P2 in human cells is about 30-50 microM, pH is close to 7.0 and the temperature is ~37C (~310K). Why have the authors measured the kinetic parameters using 293K, pH 8.0 and 400 microM F1,6P2? I could not find any information about km and/or Kd for F1,6P2 and FBPase II from Francisella tularensis throughout the paper.

The kinetic studies were performed at pH 8.0 while the crystallization was carried out at pH 6.0. Does pH have no effect on the arrangement of the FBPase II active site?

I believe that a short discussion of the above problems could clarify the concerns about the results.

Reviewer #2: RE: PONE-D-22-24612

Dear Editor,

The manuscript: New Structure of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis suggest a novel catalytic mechanism for the entire class by Abad-Zapatero et al. presents a three structures of FBPaseII from two different species with ligands. This work is aimed at elucidating the details of the catalytic reaction mechanism that is still under controversy for this class of FBPases. This paper presenting three new complexes of previously published structures, and additional results and interpretations, certainly deserves to be published. Unfortunately, this referee found significant problems with the structures, significant problems with their interpretation as well as with the proposals for the mechanism drawn. In its present form the paper is not suitable for publication.

In the referee opinion several alternative/synergistic actions need to be taken.

1) The structures need to be improved.

The structure 7txa has deeply unfinished water structure that is coupled with unexplained densities in the majority of active sites. The two 6FP refined are doubtful which problem is entangled with identification of the metal ion binding sites. The best example is provided by inspection of temperature factors of 6FP in which they vary by more than two fold in a single molecule. Two fragments of the backbone, 57-65 and 322-328 are systematically misplaced. All have weaker densities in all eight subunits. They nevertheless can be properly traced by gradual improvement of phases (adding proper elements to the model) and by gradual lowering of the contouring level of ED. The placement of phosphates in F6Ps are doubtful.

In the structure 7txb backbone is relatively well placed but contains numerous conformational errors (peptide flips) that are the most visible by comparison between subunits. Additionally I believe that they are indicators of alternative locations for 109-113 region. The subunits are widely separated and do not form any biological unit. They should be brought together for reliable modelling by crystallographic symmetries to form a biologically relevant unit. Too many water molecules at 3.7Å combined with a flipped configuration between two refined FBP (6P in place of 1P in subunit A) make the findings unreliable.

In the structure 7txg which is of the highest resolution and constitutes the basis for the entire publication the uncertainty in identification of metal ions and lack of full description of their coordination spheres makes the speculations very problematic. The best defined active site in subunit C contains most likely a product F6P intermixed/overlapping with the glycerol. My initial attempt at resolving ambiguities lead to 70% F6P and 30% glycerol. So a combination of findings in corrected 7txg and 7txb combined with E.coli structure (3d1r, that also unfortunately contains some errors, the placement of the head group of Glu213) might constitute a useful starting point for speculations about the catalytic mechanism. Unfortunately, suggesting that off-axial remote water molecule as initiating a reaction is outside of the realm of possibility, when existing literature is taken into account, and needs to be either excised or alternatively supported with solid experimental evidence.

2) More thorough interpretation based on a solid literature review need to be presented in the introduction and thrown back on a broader chemical and biochemical background that needs to be cited. It means that the discussion of the possible pathways for phosphate hydrolysis must be reviewed and cited. As a useful citation authors can use: Fundamentals of Phosphate Transfer by Anthony J. Kirby and Faruk Nome Acc. Chem. Res. 2015, 48, 1806−1814 (or any other classic works including that of the editor). This citation explains that even though a nucleophile can attack from any direction cannot be a priori excluded, the preferred one is carried out in the “in-line” direction along the connecting bond. Some experimental classic works can be cited also (Richard Honzatko or Karen Allen). Recalling the reference 4 (Brown G, Singer A, Lunin VV, Proudfoot M, Skarina T, Flick R, et al. Structural and biochemical characterization of the type II fructose-1,6-bisphosphatase GlpX from Escherichia coli. J Biol Chem. 2009;284(6):3784-92) is obviously necessary but needs to be treated with caution. This publication misrepresents the existing literature as well as misinterprets their own structure. The same mistake in my opinion have been committed by the authors of this publication. Neither structures, whether E. coli, or MT, or FT, support the notion that the water molecule is an attacking nucleophile. A much more likely nucleophile in all three species is activated Thr89/90 Oγ. Such a hypothesis should be thoroughly evaluated though, as it invokes a covalent intermediate and can be relatively easily probed. All schemes in the paper as well as most of the figures are deeply misleading as they are directed to support a very low probability pathway.

3) Alternatively the paper can be formulated as a structural report without speculating about the nature of the catalytic mechanism. However, if the authors select discussing this issues it needs to be tested. There are several tests that can be applied here. Firstly, one can test for the presence of phosphorylated Thr by use of appropriate antibodies. Secondly, one can check by isotope exchange the presence of appropriate labelled atoms (deuterium, or labeled oxygen) by many different methods (NMR, Mas Spec, fast X-ray etc). Another method is isotope exchange kinetic method. Unfortunately, the role of a referee is not to direct the necessary science, so I am only sharing some advice to guide how to achieve the publishable manuscript that would meet the scrutiny of this referee. The authors need to commit themselves to a particular solution and improve the manuscript in the spirit of the chosen direction.

In essence the paper cannot be published in its present form but may become publishable after major corrections.

Detailed remarks.

1) The paper is relatively well written and edited. However, there are many places that can be improved. For instance, I am confused with numbering and placement of individual Figures. I would recommend including Table S1 with refinement statistics inside the main body of the paper. Nowhere in the text, besides TableS1 the estimation of quality nor final statistics are mentioned.

2) The abstract must be adapted to the version of the paper authors select to publish. I absolutely agree with the proposal that the helical motif 88-94 is stabilizing the leaving phosphate and contributes to its binding but the statements about a nucleophile must be clarified or removed. Additionally arguments about activity of mutants or combination of metals must be tempered. It is very well documented that mobile loops play significant role in activity of all FBPases. Secondly, it is very well documented that different metal ions play different roles and that the inhibitory metal ion binding might be an activating factor. Multiple examples can be found in the literature. This point must be dealt with appropriate subtlety. I share the criticism of previous publications but my criticism persist towards what was written in the introduction.

3) The Figures must be reorganized to form a more logical story, it means renumber and relocate them. The Figure captions must be improved to provide more specific content. For instance, the subunit and the contouring level of the type of the ED used in the figure, must be mentioned

4) Kinetics presented in Fig2 are very interesting but unequivocally indicate that at least two metal ions are involved in catalysis as indicated by the burst phase, no matter whether only one or two types of metal ions are involved. A useful guide is also inhibitory phase that usually happens when a leaving group is associated with the separate associated metal ion. More physicochemical studies would be needed to establish the number and affinities for individual metal ion binding sites, for instance using DSC or by plasmon resonance.

5) Identification of many glycerol molecules is doubtful. Some kind of uniform test should be applied to produce more reliable identification of solute components. The claim that glycerol molecules interfere with refinement of F6P is an unjustified/unfounded technical obstacle. In Coot/Refmac it is enough to use disordered A,B,C components to obtain unreacting/nonrepelling/overlapping models representing partial occupancies.

6) Fig6B is extremely misleading. Not only placement of F6P is doubtful in form B but also the view is such that diffuses the impression that 6P is closer to the position of 1P in the substrate as defined in form B.

7) I like Fig9 in design but it needs to be improved providing that the correct ions with correct coordination spheres are depicted. See my previous doubts. Did authors used anomalous signal to detect their identities as the variable occupancy might be misleading during the refinement?

8) Fig10 presents an impossibility, at least as the chemical consensus stands currently, about the phosphotransfer reactions, and needs to be changed or deleted.

9) The Supporting information has to be edited appropriately and at present time has several small defects. For instance in Table SI2 the info about the temperature factors is muddled. Most likely the first and the last column represent minima and maxima of Bs, not as described the third and fourth value. Also RSCC cited do not provide full confidence about the proper identification of ligands. I am also abstracting from a fact that the numerical descriptions are different from the coordinate files I obtained. For instance temperature factors of Mn ions in my file of form A are 53, 145, 118, 129 which certainly are different from 53, 93, 73, 73 cited in the table. Please be consistent. I do not even mention that any attempt at refinement changes these numbers significantly.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-24612R1New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire classPLOS ONE

Dear Dr. Abad-Zapatero,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

 I asked one of the reviewers to check if they are happy with the revised version, and they suggested fixing some minor inconsistencies before it is accepted.

Please submit your revised manuscript by Jul 10 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Bostjan Kobe, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: This version of the manuscript is significantly improved in form and in spirit. It avoids major discrepancies with published knowledge. It is partially suitable for publication. Several minor problems persist that require attention and correction. It however dos not require my supervision and further review.

The most significant problem is that the paper requires careful reading and correcting a number of typos. There are multiple instances of mistyped/misplaced letters, symbols and the most particularly unintended concatenations (lack of white spaces). There are unjustified use of long hyphens and similar editing problems. I found a very puzzling use of (Ref: Nishimasu-Structure-2004). Is this a real citation? It is not on references list. Additionally I found very puzzling the statement: "This results in a C�-C� distance between the chains of 1.5 A at this position." If it is true then it is a clear error. If it is a suspected artifact then it should not be mentioned as an observation. The safest method of dealing with this issue at this stage is to remove it. It does not add anything to the body of the paper.

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Reviewer #2: No

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New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class

PONE-D-22-24612R2

Dear Dr. Abad-Zapatero,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Bostjan Kobe, Ph.D.

Academic Editor

PLOS ONE

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