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Review
Peer-Review Record

Behind the Scenes of PluriZyme Designs

Eng 2024, 5(1), 91-103; https://doi.org/10.3390/eng5010006
by Ana Robles-Martín 1,2, Sergi Roda 1,3, Rubén Muñoz-Tafalla 1,2 and Victor Guallar 1,4,*
Reviewer 1:
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Eng 2024, 5(1), 91-103; https://doi.org/10.3390/eng5010006
Submission received: 12 October 2023 / Revised: 14 December 2023 / Accepted: 22 December 2023 / Published: 3 January 2024
(This article belongs to the Section Materials Engineering)

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The Review manuscript from Robles-Martín et al. reports on the recently developed workflow for the design of PluriZymes, which are enzymes possessing at least one supplemental designed non-natural active-site.

The manuscript is generally well-written and in a hot-topic in the enzyme engineering field, but there are a list of things to fix in order to better describe the system for the generalist (and not specifically biotech specialists or protein engineers) public of the ENG Journal and for clarifying on some points. Here the list:

ABSTRACT:

- "Enzyme engineering has grown considerably in recent years, and it will continue growing" Considering this Journal covers all areas of engineering, not just biological/biochemical, I suggest to replace this phenomenalistic statement with a definition of protein engineering

- Explain better  in a phrase why enzyme engineering will represent an essential scientific solution to biopolymer degradation

- "more efficient bioprospecting" Not clear

- "Computational modeling will play a critical role in developing such solutions, allowing more efficient bioprospecting, providing structural information, and generating mechanistic studies and new hypotheses" For experts it is not necessary to explain the connection between enzyme engineering and computational modelling, but for non-expert this statement is ambiguous. Please improve it

 

1. INTRODUCTION

- "It has widely proven its importance in a vast number of applications, with a significant recent increase in the number of studies in both academia and industry." Please provide a ref. or quantify them somehow

- "along with their malle-able nature," Not clear what it is intended

- "At the experimental site" -> side

- "At the experimental site, a plethora of rational mutagenesis [2–5] and directed evolution [6–8] techniques have been at the center of the attention. We are interested, however, in the computational techniques for enzyme engineering,..."Rational mutagenesis often requires a more or less complex computational design to target specific mutations (apart from general empirical rules usually adopted in the field). Please rewrite this part to account for it

- "The pluriZyme concept is in line with recent efforts in designing protein-bearing multiple enzyme centers using different approaches "
Considering the generalist target of this Journal, it is suggested to add a figure for describing the listed approches

- "The design of pluriZymes has been based on adding catalytic triads capable of introducing hydrolytic biochemistry" -> "esterase hydrolytic biochemistry"

- "Inserting an extra active site allows having different chemistries in a single protein scaffold, reducing the costs and optimization of protein expression" Is more than one active site detrimental to protein solubility and stability? Please, you may want to explicitly add any possible detrimental (or positvie) effect in this part or in table 1, if any known

- "As such, some of our more recent designs show high activity towards the degradation of polymers, including polyethylene terephthalate (PET) nanoparticles and xylane" Please add an explicit example on how the activity on these compunds may be applied

 

 

PARAGRAPH 2

- "PluriZymes are proteins with more than one active site capable of enzymatic cataly-sis, where at least one of these has been designed by protein engineering" Redundant

- Figure 1: it is not clear if the explored binding sites (for active site introduction) are already present or are designed in concert during the exploration. Maybe the figure could be improved to include both options, as discussed in the text.

- "Therefore, adding a series of constraints to the initial structure is nec-essary, generally with a force of 5 kcal/(mol·â„«2) every 5 or 10 residues"
Not clear to a non-expert; does it mean 50 Kcal/(mol*A^2) for a 50/100 residues protein or 5 Kcal to one residue every 5 positions along the sequence?

- "If we do not find a potential new binding site, we have to consider whether we want to continue working with the system by designing a binding site"
Can you include one or more suggestions how to add a binding site?

- "Regarding this last point, we prioritize catalytic triads where we use a wild-type acidic residue, since adding negative charges to a protein might have a detrimental effect." Please cite something or explain why.

 

 

PARAGRAPH 3

- Table 1. Please remove the Journal name, as this is already included in the provided reference. Please add changes in melting temperature, optimum temperature and protein yield, if available. This will greatly enhance the general relevance of your review on designs

- "This last design is not listed in Table 1 since its manuscript is cur-rently under revision" You may want to include a preprint or the accepted manuscript in the course of this review.

 

PARAGRAPH 4

- "The latest trends regarding bioplastic recycling seek to degrade biopolymers into their monomers and transform and reuse them in new materials in a process called bio-upcycling. This concept is currently being exploited, especially for polyethylene tereph-thalate (PET)" Ok, but PET is not generally considered a bioplastic. Please explain better this section

- "One example could be PET biopolymer. The products obtained from the hydrolysis of PET are mainly ethylene glycol (EG), terephthalate acid (TPA), monohydroxyethyl ter-ephthalate (MHET), and bis (hydroxyethyl)terephthalate (BHET)" PET is not a biopolymer. Which kind of hydrolysis do the authors refer to? Enzymatic? Which enzymes?

- "For example, BHET can be reused for PET production [43–45]" You need to obtain EG and TPA for PET re-synthesis, you cannot start from BHET. Moreover, it is insane to obtain BHET as a product from biocatalyzed reactions, as usually >98% of the biodepolymerization products of PET are TPA, MHET and EG. Please fix the text

- MIL53 -> MIL-53

- "All these compounds are of industrial interest, and it would be attractive to couple the degradation process with a synthesis process in a coupled system of complex reactions"
The known big issues since '50 of the past century is not the PET degradation process, but that the starting PET monomer TPA is highly insoluble even at mM concentrations at pH < 9, that's why it is usually recovered by precipitation using this property and reused with proper solvents for PET resynthesis. This stands also for chemical depolymerized PET, which have the same problem since many decades. Do you have any suggestion to overcome this long-standing issue for enzymatic products? Please add a statement on the need to find solutions also for dealing with insoluble or low solubility compounds like PET monomer TPA (i.e, making the reaction in organic solvents or other proposals)

- "As a result, our FraC variant achieves one order of magnitude higher degradation rates than the current best PETases when operating on PET nanoparticles."
How did you compare the specific activity of a membrane bound (not in solution enzyme) to a soluble enzyme? Can you quantify the concentration of active sites in the reaction mix somehow? Please explain

 

 

CONCLUSIONS

- "Interestingly, most plu-riZymes tend to be more stable than the native proteins" It is really crucial to add information on this in table 1 (see another comment above)

- "In one transaminase design, for example, the second active site involved some residues located in a loop, in-troducing a significant stabilization effect (almost a 6 degree increase in denaturation tem-perature) [20]. The fact that our mutants are designed to interact through hydrogen bonds might also explain the high success rate, helping the successful translation of the in silico predictions into the real in vitro assays. We have designed six pluriZymes with six more to go. Only in one case did we not succeed in obtaining additional active sites: when aim-ing to introduce a second protease site in a small globular protease. The main reason for our failure seems to be the lack of an additional effective binding site(s); while some ap-parent sites were found, they did not represent a significant minimum in the substrate binding energy profile. Thus, the second step, the binding site search, might be the bottle-neck of the entire design process and a potential deal breaker.
In addition, one should consider that pluriZymes might introduce some drawbacks. Obviously, the first one is that the wild-type activity can be compromised. Moreover, the engineered artificial site has a tendency to be non-specific; additional engineering might be necessary to adapt it for specific substrates. In addition, although not frequent, the de-signed variants can introduce problems in the expression/solubility or thermal (and even pH) stability. Finally, the catalytic efficiencies of many pluriZymes we have created are far from perfect (kcal/Km ~102–103 M−1·s−1), with similar rates to the artificial enzymes. These limitations show the difficulty of adding an efficient new active site to a functional enzyme without compromising the existing chemistry." This is actually not a statement for the conclusion section. Please, add this part in a "Limitations" or similar paragraph.

Comments on the Quality of English Language

The paper English quality is generally high, with just few points that require minor fixes that can be adjusted before or after acceptance

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Your contribution is truly outstanding, providing all the necessary background information, a clear description of the algorithm, and a practical application for PET biodegradation. Well done!

Author Response

Thank you very much for your comment and valoration.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This is timely and comprehensive review on the concept of PluriZymes – engineered variants of natural enzymes that beside their native active sites also have additional ones that have been introduced into the protein structure by a rational design. A single protein scaffold of a plurizyme can catalyze different reactions, facilitating, for example, one-pot cascade reactions. Thus, the article reviews an exciting new approach to enzyme engineering that may soon attract considerable interest due to its relative simplicity and substantial success rate.

I found this review easy to read, well-structured, and documented. The concept of pluriZymes, the most efficient workflow for their development, and the examples of working pluriZymes were covered with a good style and with good use of the available literature. The text was also illustrated with four well-prepared and informative figures.

To sum up, this is a timely review on an interesting topic. No articles covering similar subjects are currently available, and I am sure that this work will attract considerable interest from the community. 

 

 

Author Response

Thank you very much for your comment and valoration.

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Thanks for the excellent work of revision. Just a couple of comments:

 

1)'''- "The latest trends regarding bioplastic recycling seek to degrade biopolymers into their monomers and transform and reuse them in new materials in a process called bio-upcycling. This concept is currently being exploited, especially for polyethylene terephthalate (PET)" Ok, but PET is not generally considered a bioplastic. Please explain better this section bio-based PET or bio-PET can be produced. Although the production of total bio-PET is not yet economically feasible, bio-PET is partially produced from renewable resources and is considered one of the most significant bioplastics (https://doi.org/10.1016/j.ese.2023.100254). However, as is not generally considered a bioplastic we could substitute the biopolymer and bioplastic
with plastic polymer."

- "One example could be PET biopolymer. The products obtained from the hydrolysis of PET are mainly ethylene glycol (EG), terephthalate acid (TPA), monohydroxyethyl ter-ephthalate (MHET), and bis(hydroxyethyl)terephthalate (BHET)" PET is not a biopolymer. Which kind of hydrolysis do the authors
refer to? Enzymatic? Which enzymes?"
Yes, we will change biopolymer into polymer.
petases -> change to ester hydrolysis'''

Yes, I agree with all, but both at technical or non-technical level there is very often a "formal" confusion on what is a bio-based polymer, a biodegradable polymer and a process with a bio-based step to hydrolyze or produce a polymer, so please avoid any possible confusion and fix the term, as it is not fixed in the reviewed version you posted. Please, add enzymatic in front of "hydrolysis": it is obvious for biotechnologists, protein engineers and similar, but the common hydrolysis that is known in the field of PET recycling is not enzymatic and when I speak with engineers and technical people, they usually think I intend chemical glycolysis or a similar process.

 

'''2)- "For example, BHET can be reused for PET production [43–45]" You need to obtain EG and TPA for PET re-synthesis, you cannot start from BHET. Moreover, it is insane to obtain BHET as a product from biocatalyzed reactions, as usually >98% of the biodepolymerization products of PET are TPA, MHET
and EG. Please fix the text The new process of recycling from BHET was pointed out by one of the Nature Catalysis reviewers (fairly new). Interestingly enough, one of our mutants stops at BHET. So it is a possibility to design enzymes that can generate BHET for recycling.'''

Ok, very interesting!

Author Response

Thank you for your kind words.

We have added the word "enzymatic" as requested in this second revision

Round 3

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Please changebiopolymer into polymer when used to mention the PET polymer. The most accepted scientific definition of "biopolymer" is a biologically (by living organisms) produced polymer. Politically this definition was extended by many istitutions, including European Union, to intend a polymer produced with non-biological steps from feedstock monomers or polymers that are at least in part from biological biomasses. In the process of PET re/upcycling you use enzymes to degrade PET and then other enzymatic or abiotic chemical and mechanical steps, but for the production of new PET the "biological step(s)" is(are) not made of raw materials that are monomers/oligomers/polymers from biomasses.

PET is not the product of any organism.

Alternatively, please provide or propose an alternative explicit definition of biopolymer

Author Response

Please see the attachment

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.


Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper could have some interest, as plurizymes may be on the arrow point of the design of enzymes bearing several active centers. Unfortunately, the current presentation is just a summary of the methods section of the previous papers, summarizing some results, and looking just a publicity advertisement of the lab, many points are explained in first person mentioning collaborations with other groups, projects, etc. This looks more appropriate for the authors´ web page than for a scientific paper.

In fact, the paper seems to give protocols to generate an artificial active center in a protein, and it is not strictly related to the concept of plurizymes, that if I understood correctly from previous papers (I have been able to find only 4 ones), consists in adding new active centers to an enzyme bearing already a native active center. They mention 8 plurizymes, while there are only 4 papers on them, and two are on the same.

The fact that among the showed “plurizymes” they select FRAC, that seems to be a structural protein where they added an active center confirms that the paper is more in generating artificial enzymes than in plurizymes. I do not understand why this artificial enzyme has not a reference, if it is so efficient is suspicious that the authors renounce to make a proper research paper on this enzyme. That in any case, it is not a plurizyme.

Thus, I do not see any interest in this presentation, as it is just a summary of the methods section of previous papers, with some aims to make publicity to the idea.

From my point of view, in the context of plurizymes, the paper could become interesting if it is greatly altered and improved. They should first discuss the advantages of having enzymes presenting several active centers (cascade reactions), there are multiple options, and some real reviews on this matter, some very recent and ignored in this paper. They should give answer to this question: Why may it be better to have two active centers in the same structure than to use two independent enzymes? Plurizymes will be among the possibilities, they should give advantages, problems and expected drawbacks of plurizymes compared to fussed proteins and other alternatives. In the Nature catalysts paper, they show that the double modified enzyme present some synergy in the electronegativity of the active centers, this important fact cannot be fully ignored, as it is a clue on the advantages of plurizymes. Then, they can explain how a plurizyme must be designed (the main topic of the current paper), compared with the design of a standard artificial enzyme, I guess that the necessity to keep the native center activity is an additional problem. Then, they can list the results where real plurizymes are developed (perhaps there are more than the 4 papers I have been able to find), leaving FRAC as an example of the potential of the developed tools to design any artificial enzyme, although I cannot find this relevant in the context of plurizymes, looks more an example that the authors are proud of, without real relation with plurizymes. And they can leave this paper only if they have a proper reference that can be accessed by the readers (even a Thesis could be valid if the authors do not have intention of publishing the results), to include results in a review without any reference seems inadequate.

A paper focused on design of standard artificial enzymes should give new information to the many papers on this regard that can be found in literature, including multiple reviews. I do not think it can have interest, and I suggest focusing on plurizymes as in this case, bur under a fully different point of way and a fully different presentation.

 

Author Response

Response to Reviewers 

Reviewer 1

The paper could have some interest, as plurizymes may be on the arrow point of the design of enzymes bearing several active centers. Unfortunately, the current presentation is just a summary of the methods section of the previous papers, summarizing some results, and looking just a publicity advertisement of the lab, many points are explained in first person mentioning collaborations with other groups, projects, etc. This looks more appropriate for the authors´ web page than for a scientific paper.

In fact, the paper seems to give protocols to generate an artificial active center in a protein, and it is not strictly related to the concept of plurizymes, that if I understood correctly from previous papers (I have been able to find only 4 ones), consists in adding new active centers to an enzyme bearing already a native active center. They mention 8 plurizymes, while there are only 4 papers on them, and two are on the same.

The fact that among the showed “plurizymes” they select FRAC, that seems to be a structural protein where they added an active center confirms that the paper is more in generating artificial enzymes than in plurizymes. I do not understand why this artificial enzyme has not a reference, if it is so efficient is suspicious that the authors renounce to make a proper research paper on this enzyme. That in any case, it is not a plurizyme.

Thus, I do not see any interest in this presentation, as it is just a summary of the methods section of previous papers, with some aims to make publicity to the idea.

From my point of view, in the context of plurizymes, the paper could become interesting if it is greatly altered and improved.

  •  They should first discuss the advantages of having enzymes presenting several active centers (cascade reactions), there are multiple options, and some real reviews on this matter, some very recent and ignored in this paper. 

Answer:

The reviewer is right, the paper is a review on our previous designs on plurizymes, and in their potential for polymer degradation. It does not aim to be a review on enzyme engineering nor cascade reactions, since it responds to an special issue on polymer degradation, and a particular invitation on how plurizymes might help in this matter.  And in order to make it a bit more appealing we try to give a more comprehensive explanation on how to perform the modeling leading to a plurizyme design (I believe significantly more comprehensive than the methods sections in our previous papers --for example giving alternatives for people not having our software, etc.). 

The definition of plurizyme is basically: an enzyme with more than one active site where at least one of them is artificially added (engineered). All the systems summarized in the paper are plurizymes based on this definition;  FraC is an octamer, thus introducing eight active sites. 

This was defined in the initial sentence in Section 2:

“PluriZymes are proteins with more than one active site capable of enzymatic catalysis, where at least one of these has been designed by protein engineering. “

 

We agree that the introduction definition might be a bit confusing and we have reformulated it. Thank you.

 

  •  Why may it be better to have two active centers in the same structure than to use two independent enzymes? Plurizymes will be among the possibilities, they should give advantages, problems and expected drawbacks of plurizymes compared to fussed proteins and other alternatives. 

[Answer]

Plurizymes offer a great deal of possibilities, beyond the simplistic approach of fussing enzymes. Currently, for example, we are inserting catalytic triads to multiple (dozens) of proteins/enzymes from a bacterial host, aiming to transform such a host into a polymer degrading bacteria.

As for cascade reactions, there are several advantages. First obviously it reduces overall protein cost. It facilitates expression costs and the necessity of optimizing different enzymes to the harsh industrial conditions. Similarly, it helps in unifying operating conditions such as optimal pH and temperature.  Moreover, the proximity of the different active sites might help in better diffusion properties and overall turnover.

This information is now more clear in the conclusion section. Thank you.

  • Then, they can list the results where real plurizymes are developed (perhaps there are more than the 4 papers I have been able to find), leaving FRAC as an example of the potential of the developed tools to design any artificial enzyme, although I cannot find this relevant in the context of plurizymes, looks more an example that the authors are proud of, without real relation with plurizymes. 
  • And they can leave this paper only if they have a proper reference that can be accessed by the readers (even a Thesis could be valid if the authors do not have intention of publishing the results), to include results in a review without any reference seems inadequate.

 

[Answer]

As mentioned  FRAC  has eight active sites in the final active configuration of the pore complex. The FRAC paper, that includes two of the PluriZymes in table 1, is in a third review stage in Nature Catalysis (where only 1 of 5 reviewers has asked for some extra experiments). We expect acceptance of it within a few weeks. Actually, as agreed with the editors, this Eng review paper will only appear after acceptance of the FRAC Nature Catalysis one. 

The only system that will not be published is the xylanase one. We will remove it from Table 1, which will only include now systems with peer review publications. Still  we will mention it in the main text clearly stating that the manuscript is in process, as we believe it is a very interesting  outcome depicting the possibility of further mixing different biochemical processes.

 

A paper focused on design of standard artificial enzymes should give new information to the many papers on this regard that can be found in literature, including multiple reviews. I do not think it can have interest, and I suggest focusing on plurizymes as in this case, bur under a fully different point of way and a fully different presentation.

Answer:

With two Nature Catalysis (one of them chosen as cover) and one Angewandte Chemie publications within 2 years , we believe that pluriZymes are one of a kind enzymes. Moreover, as we state in the paper, they are not difficult to make, with ~90% success rate. Thus, we do believe it is of the general interest to dig more into how to make them and to publicize their potential. This is not a review paper on enzyme engineering but on pluriZymes. Please notice that we already find a very recent review largely centered on pluriZymes from another lab, mdpi.com/1422-0067/23/10/5304#B157-ijms-23-05304,  which also encouraged us to make this manuscript.

As stated, we have modified the conclusion section to stress more on these points and the potential of pluriZymes, thank you.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer:

The authors summarize the recent development in PET nanoparticles degradation and comprehensive recipe for developing the pluriZyme. It would have been nice to have a little more emphasis on/introduction to the basic biocatalysis (for example, what are the advantage of using pluriZyme to biocatalytic application and the requirements to have an efficient cascaded catalysis and why?). As it stands, the article will be of most interest to practitioners in the field, serving as a snapshot of what has been achieved so far in the field and what is the current state-of-the-art. The article will be a fine contribution as it stands, but any effort to connect more closely to the underlying bioengineering and biocatalysis would be appreciated if the authors can do that.

1) There are only few references providing the bioengineering and its applications. If it is possible to add the contents about substrate channeling with another three or four reference that would be ideal given the contents of rosseta in the text you provided. 

Comments for author File: Comments.pdf

Author Response

The authors summarize the recent development in PET nanoparticles degradation and comprehensive recipe for developing the pluriZyme.

It would have been nice to have a little more emphasis on/introduction to the basic biocatalysis (for example, what are the advantage of using pluriZyme to biocatalytic application and the requirements to have an efficient cascaded catalysis and why?). 

 

[Answer]

Thank you for your suggestion and support for our manuscript. As stated to the first reviewer:

 

Plurizymes offer a great deal of possibilities, beyond the simplistic approach of fussing enzymes. Currently, for example, we are inserting catalytic triads to multiple (dozens) of proteins/enzymes from a bacterial host, aiming to transform such a host into a polymer degrading bacteria.

As for cascade reactions, there are several advantages. First obviously it reduces overall protein cost. It facilitates expression costs and the necessity of optimizing different enzymes to the harsh industrial conditions. Similarly, it helps in unifying operating conditions such as optimal pH and temperature.  Moreover, the proximity of the different active sites might help in better diffusion properties and overall turnover.

This information is now more clear in the conclusion section. Thank you.

 

1) There are only few references providing the bioengineering and its applications. If it is possible to add the contents about substrate channeling with another three or four reference that would be ideal given the contents of rosseta in the text you provided. 

 

[Answer]

Yes, we believe it is a good idea. Several references have been added in this context at the “Conclusions” section:

 

“Moreover, it can ensure that both catalytic active sites are nearby, potentially accelerating the total reaction rate through substrate channeling [42-45]”

Reviewer 3 Report

Comments and Suggestions for Authors

In this review, the authors summarized recent advance in the design of pluriZyme, which may improve the catalytic performance or perform a cascade reaction by adding a second (or even more) active site(s) in an existing enzyme or protein. For this aim, they presented many valuable experiences, the software, catalytic distances and so on. Meanwhile, the successful designs of pluriZymes in authors’ lab demonstrated the efficiency of the recipe,especially pluriZymes in PET nanoparticles degradation to promote the circular economy.

It is an interesting review, and I recommend acceptance of this manuscript after the following issues are addressed.

1) Some important terms need to be explained clearly in the introduction, such as enzyme engineering and the clarification of the relevance of enzyme engineering and pluriZyme.

2)The challenges and perspective of pluriZymes should be supplied in the conclusion part.

3) Experimental verification is not explained in part 2.4 “Refinement and Experimental Validation”

4) Table 1 did not appear in the text.

5) Abbreviations of proper nouns appearing for the first time shall be marked with full names, such as PET.

Author Response

Reviewer 3

In this review, the authors summarized recent advance in the design of pluriZyme, which may improve the catalytic performance or perform a cascade reaction by adding a second (or even more) active site(s) in an existing enzyme or protein. For this aim, they presented many valuable experiences, the software, catalytic distances and so on. Meanwhile, the successful designs of pluriZymes in authors’ lab demonstrated the efficiency of the recipe,especially pluriZymes in PET nanoparticles degradation to promote the circular economy.

It is an interesting review, and I recommend acceptance of this manuscript after the following issues are addressed.

1) Some important terms need to be explained clearly in the introduction, such as enzyme engineering and the clarification of the relevance of enzyme engineering and pluriZyme.

  • Explain enzyme engineering
  • Clarify the relevance of enzyme engineering
  • Clarify the relevance of plurizyme

Answer:

Thank you for your recommendations and support of our manuscript. Those terms have been better clarified in the introduction and conclusion section.

 

2)The challenges and perspective of pluriZymes should be supplied in the conclusion part.

 

Answer:

Thank you for your suggestion. We have expanded the conclusion section with few more challenges and current research.

 

3) Experimental verification is not explained in part 2.4 “Refinement and Experimental Validation”

  • Explain the Experimental Validation

Answer:

Yes, it should only be Refinement. It is the recipe for the computational workflow. We have modified it; thank you for seeing it.

 

4) Table 1 did not appear in the text.

Answer:

Thank you, we have added reference to Table 1

 

5) Abbreviations of proper nouns appearing for the first time shall be marked with full names, such as PET.

Answer:

Thank you. we have revised them. 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I already mentioned in my previous report that in my opinion the concept and development of plurizymes is among the most important in the area of biocatalysts design.

However, I am afraid that the new version of the manuscript has scarce improvements compared to the previous one. Still, there are too many first person uses in the explanations and the new information or discussion compared to the previous publications are not significant. It remains more publicity than science advances. They mention review literature on generating proteins bearing multiple enzyme centers, but still they do not cite them in the paper. And the advantages / drawbacks of plurizymes (even being a very nice and interesting new concept) are not discussed. In a review, problems should be mentioned, as they are in some of the previous reviews, it is not a demerit of this outstanding idea, but must be recognized as a possible problem. Also, compare this idea with chimeric enzymes and other strategies will give this review paper a higher interest.

That way, an improved version of the paper focused on plurizymes should be highly interesting,

However, the paper is focused on the development of a new artificial enzyme that the authors try to pass for a plurizyme when it is a standard artificial enzyme.  It is because the special issue is on a product degradation that is recognized by the enzyme.

I have several problems with consider this enzyme as a “plurizyme”:

Taken from their previous publication, this was the definition of plurizyme in the paper:

“For this purpose, we used recently published computational tools to design PluriZymes, the name we assigned to the concept of adding multiple active sites to the same enzyme structure.

That is, previous plurizymes always added an additional active center to a previously existing one, doting the enzyme of a new and different active center. This makes that the building of plurizymes is more difficult than the building of a standard artificial an enzymes, as not only yhe new active center must work, also the native one must be preserved. This is not remarked enough in the paper.

They argue that as the protein where they introduce the active center is multimeric, they introduce multiple active centers. The difference with previous plurizymes, where the name  distinguish the new idea from standard artificial enzymes, is that they introduce just one active center by monomer in a no catalytically functional protein. In the previous Nature catalysts they use a dimeric enzyme (bearing in native state two active centers, but identical) and only call it plurizyme when they introduce a second active center in the monomer. That makes that the plurizyme has two different active centers with different properties, not several identical copies of the same active center. As they make enzymes with several functions, they can call the new enzyme a “plurizyme”. The enzyme where they focus the attention has multiple copies of the same active center, and as most multimeric enzymes, the active centers are identical and are the only function of the enzyme. This means that it is a genetically engineered multimeric enzyme, the difficulties here very likely are in keeping the association  capacity, but  it is not a plurizyme as the other ones. In fact, in title, the word “plurizyme” is not used:

Robles-Martín, A., Amigot, R., Fernandez-Lopez, L., Roda, S., Alcolea-Rodriguez, V., Heras-Márquez, D., Gonzalez-Alfonso, J.L., Coscolín, C., Almendral, D., Plou, F.J., Portela, R., Bañares, M. A., Martínez-del-Pozo, A., García-Linares, S., Ferrer, M., Guallar, V.. Design of pore-based nanoreactors for nano-polyethylene terephthalate depolymerization. Nature Catalysis

While in the other papers, this word is in title (except when the launched the term).

Therefore, this is a standard artificial enzyme, not an enzyme bearing several DIFFERENT active centers, and is not in the category of plurizyme.

As they have not submitted a copy of the paper version in evaluation, it is hard to see how the paper treat this, as it is in evaluation, it is hard to know if really the paper is near to be accepted or not. My experience in Nature catalysis is that 1:4 is not a guarantee of final acceptance if the matter raised by the negative referee are serious and well defended by the referee. I understand the agreement with the editor of this journal to delay the publication until the Nature catalysis submission is accepted, but copyright issues may rise if the figures are identical in the original paper and in this review. Moreover, if the paper has not been accepted after more than one month, it is not unlikely that the paper may be finally rejected.

In any case, in my opinion, if they want to keep the focus on the new enzyme, title and introduction should be changed to artificial enzymes, and plurizymes may be consider one of the examples achieved using the methodology (in fact plurizymes are a kind of artificial enzymes). Perhaps they can include in title the program or strategies used for the design of these artificial enzyme.

Minor point:

The references for no published papers should have details as “accepted for publication”, “in press” (in this case including doi) or submitted (as it is the case of the paper that is used as main example). A submitted paper may be no finally published. They have a couple of papers with publication date (that is not possible to give if the paper is not already published) and without any other detail that are in these categories.

Author Response

Answer to reviewer 1

...They mention review literature on generating proteins bearing multiple enzyme centers, but still they do not cite them in the paper.

Response. Accordingly, we have added a section in the intro

“…to perform a cascade reaction [17,18]. The pluriZyme concept goes in line with recent efforts in designing protein bearing multiple enzyme centers using different approaches: i) self-assembly of histidine-tyrosine peptides mimicking a catalytic microenvironment [19] ; ii) using a noncatalytic protein scaffold (a lactococcal multidrug resistance regulator) resulting in two abiological catalytic sites [20,21]; iii) coupling of metal nanoparticles to gain catalytic properties [22-25]; iv) cofactor modification for a new host that incorporated both a mimic of NADH and a flavin analog [26].”

 

...And the advantages / drawbacks of plurizymes (even being a very nice and interesting new concept) are not discussed. In a review, problems should be mentioned, as they are in some of the previous reviews, it is not a demerit of this outstanding idea, but must be recognized as a possible problem. Also, compare this idea with chimeric enzymes and other strategies will give this review paper a higher interest.

Response: Now the conclusion section includes a paragraph about potential drawbacks of designing pluriZymes.

“…The main reason for our failure seems to be the lack of an additional effective binding site(s); while some apparent sites were found, they did not represent a significant minimum in the substrate binding energy profile. Thus, the second step, the binding site search, might be the bottleneck of the entire design process and a potential deal breaker.

In addition, one should consider that pluriZymes might introduce some drawbacks. Obviously, the first one is that the wild-type activity can be compromised. Besides, the engineered artificial site has a tendency to be non-specific; additional engineering might be necessary to adapt it for specific substrates. In addition, although not frequent, the designed variants can introduce problems in the expression/solubility or thermal (and even pH) stability. Finally, the catalytic efficiencies of many pluriZymes we have created are far from perfect (kcal/Km ~ 102-103 M-1s-1), with similar rates to the artificial enzymes. These limitations show the difficulty of adding an efficient new active site to a functional enzyme without compromising the existing chemistry.“

 

I have several problems with consider this enzyme as a “plurizyme”:

This makes that the building of plurizymes is more difficult than the building of a standard artificial an enzymes, as not only yhe new active center must work, also the native one must be preserved. This is not remarked enough in the paper.

Yes, after reading your review we must admit that we agree with your point of view. Thus, we have changed pretty much the entire polymer section. We do not refer to fraC anymore as a pluriZyme but as a polymer degrading engineered protein that we obtained using exactly the same technology as the one used in pluriZyme designs. Thank you for insisting in this point!

Moreover, we have expanded the section with additional efforts in this line.

As they have not submitted a copy of the paper version in evaluation, it is hard to see how the paper treat this, as it is in evaluation, it is hard to know if really the paper is near to be accepted or not. My experience in Nature catalysis is that 1:4 is not a guarantee of final acceptance if the matter raised by the negative referee are serious and well defended by the referee. I understand the agreement with the editor of this journal to delay the publication until the Nature catalysis submission is accepted, but copyright issues may rise if the figures are identical in the original paper and in this review. Moreover, if the paper has not been accepted after more than one month, it is not unlikely that the paper may be finally rejected.

In any case, in my opinion, if they want to keep the focus on the new enzyme, title and introduction should be changed to artificial enzymes, and plurizymes may be consider one of the examples achieved using the methodology (in fact plurizymes are a kind of artificial enzymes). Perhaps they can include in title the program or strategies used for the design of these artificial enzyme.

 

Response: Again, we agree and we have removed the fraC paper from the references (and table1). In addition, the second in review paper, the J Phys Chem B one, has been accepted so we can now include it as a full reference in a few days.

 

As for the Nature Catalysis paper, we are still in the third review stage. The reviewer asked us about the degrading mechanism of fraC and that forced us to do extensive experiment. We now have the mechanism worked out for both mutants and we will submit the answer by sometime next week. In any case, we have removed the reference from the manuscript and just mentioned that it is in third review and also that the results were presented already in a conference. In this way we can uncouple these two papers.

We really want to thank you for your overall reviewing.

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The main problem has been solved, as they did not call pluryzyme to Frac. The strategy used by the authors is very good, they focus in pluryzymes most of the paper and Frac is not called plurizyme although is mention that has been prepared following similar approaches.  I am unsure that really the new information or discussions on this review are a significant advance, but it is clear that the editor thinks it is.

However, some points still should be considered:

.- The paper still is like a advisement of the strategy and the group, with continuous references to projects and collaborations (that should be just in acknowledgments section, with the strength that they think they should be given, but they have no interest for the readers of the paper)

.- In enzymes bearing several active centers there are some recent reviews, mentioned in the first report, and that remain without being cited. Moreover, in the list of strategies to design enzymes bearing two active centers, they forget the one that is more “similar” to a plurizyme, the fused proteins.

.- The problems of plurizymes that I want to be commented are extended to all enzymes bearing several active centers, and they are listed in the review in IJMS mentioned in previous report: difficulties to adjust the ratio of catalytic active centers to optimize the reactions, possible different stabilities of both catalytic active centers.

.- I think that they can make more emphasis in the difficulties (that is a merit of the plurizymes)  of adding a new active center trying to keep “intact” the other active center.

.- The references to submitted or accepted papers should be maintained, just the citation should be in a correct way (without year, and showing the status of the paper).

I fully disagree with eliminating the citation to the submitted paper to Nature catalysts, just should include just a “submitted” in the reference, without date. I still have problems in accepting that we cannot see the “submitted” paper, as copy-right issues can raised. And the paper has been in revision for months, it is not so sure that the paper will be finally accepted, however, the editor has accepted this situation and I will go not deeper on this.

The strategy of no calling plurizyme the Frac enzyme is good. However, the section heading still call the enzyme a plurizyme, it may be better to use two headings, the first one can maintain the title and discuss the efforts to add a second active center to PETasas, and a second one showing the alternative solution using the FRAC, but naming this artificial enzyme, or whatever they want.

Why did they delete the mutations used to produce the catalytic FraC in this version?  They should be added, the readers of the review should not need to check the original paper for this critical information. This is most strange.

This should be in references sections, properly cited:

These results have already been presented at the 2022 Girona Seminars (https://www.gironaseminar2020.com) and are currently under the third revision in Nature Catalysis.

 

Author Response

Before making the final changes to ammend the last concerns of the reviewer, I would like to ask him to choose between these two options (we are fine with either one):

1) We keep the manuscript as it is, without information of the fraC mutants, and removing it from Table 1. This should allow immediate publication

2) We go back to the previous format, adding all info for the fraC paper, but then we would need to wait until the Nat Cat  acceptance before its publication.

We just submitted last week the third review in the Nature Catalysis manuscript. I am uploading the submitted manuscript here as well (instead of our revised one ) for the reviewer consideration

 

 

Author Response File: Author Response.pdf

Round 4

Reviewer 1 Report

Comments and Suggestions for Authors

I think that the best option is to wait for the acceptance of the NC paper and makes a complete paper (together to the other considerations on my previous report). I understood from previous versions that the editor already agreed to wait to publish this paper to have accepted the NC paper, and I think this is the best option.

After no calling calling FRAC a plurizyme, the main concern was solved (although remember to place FraC in a specif heading, not in the leading of plurizymes for degrading PET). The other are formal or minor scientific points.

Now, I can confirm from the NC whole paper, that this is just an artificial enzyme, no a plurizyme.

Author Response

Reviewer response

Dear Reviewer, in response to your last revision we have added the information on the double mutant and the citation to the Nature Catalysis manuscript (to be updated once accepted, we just heard back from the third review iteration in Nature Catalysis and it is now reduced to some minor revision requested by only 1 (out of 5) reviewers. So we believe that the manuscript will be accepted in 2-3 weeks.). We have also corrected the heading  of the 4th section, now it reads:

“4. PluriZymes and related designs for polymer degradation”

 

In addition, the IJMS review is now highlighted (along with the concept of fused proteins) in the manuscript and we have emphasized the difficulties in adding a second active site without affecting the main one.

Thank you for your suggestions that certainly made the paper better!

Round 5

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have followed most of my suggestions, perhaps they can extend on the problems of having two active centers, perhaps with different stability and activity, but I think that 4 versions are enough

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