From the journal RSC Chemical Biology Peer review history

13-Sep-2020

Dear Dr Czekster:

Manuscript ID: CB-ART-08-2020-000142
TITLE: Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports. Although one reviewer feels that the work is not in scope for the journal, I invite you to address these concerns in a revision. The suggestions by the other reviewers also indicate that major revisions are necessary.

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Associate Editor, RSC Chemical Biology

************

Reviewer 1

Harding et al. described the characterization of BtCDPS, a Cyclodipeptide synthases from Bacillus Thermoamylovorans. The paper features two crystal structures, the analysis of tRNA isoaceptors, misacylated tRNAs to explore alternative amino acids as substrates (Ile, Met, Val, unnatural amino acids), the synthesis and screening of alternatively activated amino acids leading to the identification DBE as an alternative (and much smaller) activation group, and modelling of the reaction/activation step, which was further supported by intact MS. Studying the first thermostable CDPS is a nice add-on.
The work is technically sound. The main limitation is that 2,5 diketopiperazines can be made much easier by classical organic synthesis – with a much broader scope. As such, the findings by Harding et al. are certainly of basic interest for the understanding of CDPS and related enzymes but they do not address or improve a pending biotechnological problem or limitation. Therefore, the manuscript fails to meet the high requirements for RSC Chem Biol. Organic & Biomolecular Chemistry would be an appropriate journal for this work.

Reviewer 2

The manuscript by Harding et al. is about a class of enzymes called cyclodipeptide synthases (CDPS) that catalyse the formation 2,5-diketopiperazine-containing natural products. These enzymes generally employ aminoacylated-tRNA as activated substrates for formation of cyclic dipeptides. In this report, the authors demonstrate the potential of using dinitrobenzyl-activated amino acids as an alternative substrate by one of the enzymes in this class, BtCDPS. This is an interesting finding. Few highlights of the paper along with some concerns are as below:

1. The authors of this manuscript have solved the crystal structure of Bacillus thermoamylovorans cyclodipeptide synthase enzyme, BtCDPS (apo form). This enzyme catalyses the synthesis of cyclo(L-Leu-L-Leu). The structure of BtCDPS described in this report is very similar to other cyclodipeptide synthase enzymes, including yvmC-BLIC, AlbC, and Rv2275. Using this crystal structure, the authors propose that Arg153 might play an important role in binding to the phosphate backbone of the tRNA. However, the co-crystal structure of tRNA:CDPS would be required to confirm this hypothesis. Are there any suggestions of the role of this arginine in another recent study on the same class of enzymes (RNA, 2020, DOI: 10.1261/rna.075184.120.)?
2. The authors then investigated the ability of 5 leu-tRNA isoacceptors to form cyclo(L-Leu-L-Leu) using an in vitro end point assay. All these tRNA yielded similar amounts of cyclo(L-Leu-L-Leu). It was also demonstrated that BtCDPS can accept a few other natural amino acids and two unnatural Leu derivates, cyclo-pentaalanine, trifluoroleucine as substrates.
3. Additionally, it was shown that wt and S33C mutant of BtCDPS have similar activity. This is an interesting result as S33 is involved in the catalytic cycle.
4. In order to find a minimalistic substrate for BtCDPS, the authors first tested the binding affinity of Leu-PANS to BtCDPS using ITC and no binding was observed till 2.5 mM concentration of Leu-PANS. I could not find the ITC data- it might be good to include this data in the supporting information. This led authors to conclude that the ester bond is important for activity of BtCDPS. I am not entirely convinced by the arguments presented in this section. One would expect Leu-PANS to bind the BtCDPS but due the amide bond linking the amino acid to the ribose sugar, the reaction would not go any further. I think the authors would need to present a better argument here and support it using additional data. Here is one suggestion: if Leu-PANS can inhibit the reactions of Leu-tRNA and Leu-DBE substrates, it would support the hypothesis that Leu-PANS does not bind to the active site of BtCDPS.
5. The authors then tested Leu-umb, Leu-PNP, Leu-DBE as substrates for BtCDPS. Leu-DBE appears to be a substrate for BtCDPS. This is the most important finding of the paper but the authors do not give a quantitative comparison (of product formation) between Leu-DBE and tRNA as substrates. Comparing Figure 3B with Figure 2A, I think this would be ~12:400, i.e ~3% (for S33C mutant). I think this is relatively low but might be a good starting point for directed evolution for generation of better mutants of BtCDPS.
6. Computational docking studies: The DBE-Leu substrate was then docked into the apo-BtCDPS. These studies predicted that DBE-Leu binds well to the P1 pocket of the enzyme but not so well to the P2 pocket.
7. Intact protein mass spectrometry showed that the presence of trapped acyl-enzyme intermediate supporting the computational studies


Good about the paper:
- Lot of data is presented in the manuscript.
- Good structural characterisation of apo-BtCDPS, along with comparison of other known structures in this class of enzymes.
- Several aspects of BtCDPS are studied: tRNA tolerance, amino acid tolerance, activity of S33C mutant.
- Proposal that DBE-aa can be a substrate for this class of enzyme.
- Intact MS data to show the presence of acyl-enzyme intermediate.

Major concerns:
- Would it be possible to use Leu-DBE as substrate for this class of enzymes for any practical application? Any argument along these lines would increase the impact of the manuscript. One advantage that I can see for using aa-DBE substrate is that the synthesis of 2,5-diketopiperazine products is no longer dependent on a two-step mechanism. For example, synthesis of non-natural 2,5-diketopiperazine products will no longer be dependent on aminoacyl tRNA synthetase charging the unnatural amino acid onto the tRNA. Of course, synthesis of such non-natural 2,5-diketopiperazine products would require directed evolution of new CDPS.
- Section on Leu-PANS would need major changes.

Minor concerns:
- I could not find the structure of S33A mutant in Figure 1, mentioned on page 3- “The crystal structure of full-length (residues 1-232) and S33A mutant enzymes was solved at a resolution of 1.69 Å and 1.78 Å, respectively (Fig. 1)”.
- Figure 1: It might be good to mention that the structure has 1,2-hexanediol (in pink).
- Figure 3C: “The amount of DBE-OH linearly increased with increasing enzyme concentration (Fig. 3C)”. Are there only three points that are used to make a straight line? The authors would need more data points.
- It might be good to move Figure 3d after Figure 4.

Few other suggestions:
1. The suggestion that Arg153 might plays an important role in binding to the phosphate backbone of the tRNA was interesting. Given that Leu-DBE does not have the phosphate group. It might be good to test the activity of Arg153A (or L) mutants with Leu-DBE substrate.
2. It will be interesting to see if Leu-PANS can inhibit the activity of the enzyme.

Reviewer 3

The authors report a piece of interesting work about Bacillus Thermoamylovorans cyclodipeptide synthases (BtCDPS), which can produce cyclic dipeptide products by coupling two aminoacylated tRNA substrates. The authors solved the structure of BtCDPS, explored its specificity toward different amino acid substrates, and particularly find a very simple start molecue, leucine dinitrobenzyl ester (Leu-DBE), instead of aa-tRNA as a substrate, which can largely facilitate the synthesis of cyclodipeptide. The conclusion are well supported by the experimental results. But I still have some concerns as below.
1) The kinetic curves of Leu-DBE cyclization catalyzed by BtCDPS and Leu-DBE hydrolysis are lacking.
2) The yield of Leu-DBE cyclization catalyzed by wt BtCDPS or even the S33C mutant is very low (1% or 2%). And the active amino ester is very easy to hydrolyze in the buffer. I am concerning about the improvement capacity of this enzyme.
3) The authors mentioned BtCDPS is a thermostable enzyme. What is the activity of this enzyme toward different substrates at higher temperature?

Response to Reviewers


We thank the reviewers for their time and skill in assessing our work, and provide a point-by-point response (marked with ####) below. Text added to the main manuscript is highlighted in quotation marks. We have also highlighted alterations in red on the manuscript (Marked changes).



Reviewer #1 (Remarks to the Author):

Harding et al. described the characterization of BtCDPS, a Cyclodipeptide synthases from Bacillus Thermoamylovorans. The paper features two crystal structures, the analysis of tRNA isoaceptors, misacylated tRNAs to explore alternative amino acids as substrates (Ile, Met, Val, unnatural amino acids), the synthesis and screening of alternatively activated amino acids leading to the identification DBE as an alternative (and much smaller) activation group, and modelling of the reaction/activation step, which was further supported by intact MS. Studying the first thermostable CDPS is a nice add-on.
The work is technically sound. The main limitation is that 2,5 diketopiperazines can be made much easier by classical organic synthesis – with a much broader scope. As such, the findings by Harding et al. are certainly of basic interest for the understanding of CDPS and related enzymes but they do not address or improve a pending biotechnological problem or limitation. Therefore, the manuscript fails to meet the high requirements for RSC Chem Biol. Organic & Biomolecular Chemistry would be an appropriate journal for this work.

#### We thank the reviewer for this general evaluation of our work and appreciate the acknowledgement that the work is technically sound. We would like to point out that our manuscript does not claim to offer a new method to produce cyclic dipeptides. Instead, we highlight the discovery, for the first time, of an enzyme of this class catalysing peptide bond formation using a non-aminoacylated tRNA as substrate. Our work is the first step towards future expansion and discovery of minimalistic substrates for CDPS enzymes, and provides a rationale for substrate selection and usage. We also performed experiments exchanging the “tRNA body” of a substrate, and this uncoupling of specificity for tRNA and specificity for the amino acid revealed key insights into how CDPS enzymes select substrates, which can be further exploited in future work.
Although our work is not focused on biocatalysis to produce a cyclic dipeptide, it important to point out that despite the fact that these molecules can be easy to produce synthetically in some cases, 5 mg of cLL costs £318 (https://www.scbt.com/p/cyclo-leu-leu-952-45-4 Santa Cruz Biotech - sc-207464), and that BtCDPS is predicted to participate in the biosynthesis of the natural product pulcherrimin, which is not commercially available.


Reviewer #2 (Remarks to the Author):

The manuscript by Harding et al. is about a class of enzymes called cyclodipeptide synthases (CDPS) that catalyse the formation 2,5-diketopiperazine-containing natural products. These enzymes generally employ aminoacylated-tRNA as activated substrates for formation of cyclic dipeptides. In this report, the authors demonstrate the potential of using dinitrobenzyl-activated amino acids as an alternative substrate by one of the enzymes in this class, BtCDPS. This is an interesting finding. Few highlights of the paper along with some concerns are as below:

#### We thank the reviewer for their appreciation of our work.

1. The authors of this manuscript have solved the crystal structure of Bacillus thermoamylovorans cyclodipeptide synthase enzyme, BtCDPS (apo form). This enzyme catalyses the synthesis of cyclo(L-Leu-L-Leu). The structure of BtCDPS described in this report is very similar to other cyclodipeptide synthase enzymes, including yvmC-BLIC, AlbC, and Rv2275. Using this crystal structure, the authors propose that Arg153 might play an important role in binding to the phosphate backbone of the tRNA. However, the co-crystal structure of tRNA:CDPS would be required to confirm this hypothesis. Are there any suggestions of the role of this arginine in another recent study on the same class of enzymes (RNA, 2020, DOI: 10.1261/rna.075184.120.)?

#### To address this point raised, we cloned, expressed, purified and characterized a mutant of R153 (BtCDPSR153A). This mutant was catalytically inactive, demonstrating the crucial role it plays in the reaction. Our WT structure showed Arg153 in an inward facing conformation, interacting with the active site serine residue and in a prime position to be directly involved in substrate binding and positioning for catalysis. The crystal structure for the mutant BtCDPSR153A highlights its importance in correctly positioning the active site Ser33 sidechain. The conformation of Ser33 sidechain is rotated by ~85°, which orientates it to an inward facing position and closer to Tyr199 (forming a new H-bond) in the structure of BtCDPSR153A when compared to Wt BtCDPS (2.8 Å vs 3.5 Å). These new findings have been reported in the manuscript in the following paragraph:
“ To validate the docking experiment and test the importance of R153 to reactivity and active site architecture, we produced the mutant R153A (BtCDPSR153A). Structurally BtCDPSR153A is extremely similar to the Wt BtCDPS and BtCDPSS33C. However, activity with both Leu-tRNALeu and Leu-DBE is completely lost, supporting our observation that this residue likely has an important role during catalysis. Besides, the loss of enzyme-substrate interactions, the structure of BtCDPSR153A highlights R153 importance in correctly positioning the active site Ser33 sidechain (Fig. S13). The conformation of S33 sidechain is rotated by ~85°, which orientates it to an inward facing position, closer to the sidechain of Y199 (another catalytic residue) to form an alternative H-bond in the structure of BtCDPSR153A when compared to wild type BtCDPS (2.8 Å vs 3.5 Å). Additionally, helix-7 is displaced (<2 Å) in the BtCDPSR153A, due to the absence of R153.”

Furthermore, prior to our work arginine residues had been hypothesized to be involved in substrate binding. Previous work from Bonnefond et al1 had produced a similar mutation, but in that case only a moderate effect on activity was observed, while no structure was available for that particular mutant.

2. The authors then investigated the ability of 5 leu-tRNA isoacceptors to form cyclo(L-Leu-L-Leu) using an in vitro end point assay. All these tRNA yielded similar amounts of cyclo(L-Leu-L-Leu). It was also demonstrated that BtCDPS can accept a few other natural amino acids and two unnatural Leu derivates, cyclo-pentaalanine, trifluoroleucine as substrates.

3. Additionally, it was shown that wt and S33C mutant of BtCDPS have similar activity. This is an interesting result as S33 is involved in the catalytic cycle.

#### As suggested by another reviewer we have since added rates for reactions conducted with S33C and WT enzyme, as well as end point assays (Fig. 3 D & E, S21).

4. In order to find a minimalistic substrate for BtCDPS, the authors first tested the binding affinity of Leu-PANS to BtCDPS using ITC and no binding was observed till 2.5 mM concentration of Leu-PANS. I could not find the ITC data- it might be good to include this data in the supporting information. This led authors to conclude that the ester bond is important for activity of BtCDPS. I am not entirely convinced by the arguments presented in this section. One would expect Leu-PANS to bind the BtCDPS but due the amide bond linking the amino acid to the ribose sugar, the reaction would not go any further. I think the authors would need to present a better argument here and support it using additional data. Here is one suggestion: if Leu-PANS can inhibit the reactions of Leu-tRNA and Leu-DBE substrates, it would support the hypothesis that Leu-PANS does not bind to the active site of BtCDPS.

#### We thank the reviewer for this thoughtful suggestion. We have tested Leu-PANS as an inhibitor for the reaction and did not observe inhibition when Leu-PANS was present at concentrations as high as 2mM. Figure S21.B illustrates that Leu-PANS does not affect the rate of BtCDPS reaction, thus suggesting Leu-PANS does not bind to BtCDPS. Furthermore, we included the ITC data (Figure S19) in which no binding was observed. We therefore stand by our initial rationalisation that the ester bond is important for binding, which is now supported by this additional experiment. We modified the text to include this additional experiment:
“We hypothesised Leu-PANS would interact with the substrate binding pocket. However, ITC experiments (at concentrations as high as 2 mM) showed no binding occurred (Fig. S19). In addition, we tested Leu-PANS as an inhibitor in the aminoacylated tRNA assay, but did not observe any inhibition of BtCDPS activity (concentrations up to 2 mM) (Fig. S21), supporting our ITC experiments. We predict the ester bond linking the amino acid to the nucleotide is playing an essential role in substrate selection in CDPS enzymes.”

5. The authors then tested Leu-umb, Leu-PNP, Leu-DBE as substrates for BtCDPS. Leu-DBE appears to be a substrate for BtCDPS. This is the most important finding of the paper but the authors do not give a quantitative comparison (of product formation) between Leu-DBE and tRNA as substrates. Comparing Figure 3B with Figure 2A, I think this would be ~12:400, i.e ~3% (for S33C mutant). I think this is relatively low but might be a good starting point for directed evolution for generation of better mutants of BtCDPS.

#### We thank the reviewer for this suggestion and added information about reaction yield when reaction is performed with DBE-Leu and aminoacylated tRNA. As the reviewer points out this is a good starting point to create better substrates, building on this framework. A comparison, and acknowledgement of the significantly less efficient Leu-DBE substrates has been noted in the manuscript:

“Alongside identifying a minimal substrate (albeit less efficient than natural aa-tRNA)”
“(78 M cLL product formed per M Wt BtCDPS with a Leu-tRNALeu substrate, compared to 0.8 or 2.4 M cLL product formed per M Wt or S33C variants, respectively),”

6. Computational docking studies: The DBE-Leu substrate was then docked into the apo-BtCDPS. These studies predicted that DBE-Leu binds well to the P1 pocket of the enzyme but not so well to the P2 pocket.

7. Intact protein mass spectrometry showed that the presence of trapped acyl-enzyme intermediate supporting the computational studies


Good about the paper:
- Lot of data is presented in the manuscript.
- Good structural characterisation of apo-BtCDPS, along with comparison of other known structures in this class of enzymes.
- Several aspects of BtCDPS are studied: tRNA tolerance, amino acid tolerance, activity of S33C mutant.
- Proposal that DBE-aa can be a substrate for this class of enzyme.
- Intact MS data to show the presence of acyl-enzyme intermediate.

#### We thank the reviewer for appreciating these positive aspects of our work.

Major concerns:
- Would it be possible to use Leu-DBE as substrate for this class of enzymes for any practical application? Any argument along these lines would increase the impact of the manuscript. One advantage that I can see for using aa-DBE substrate is that the synthesis of 2,5-diketopiperazine products is no longer dependent on a two-step mechanism. For example, synthesis of non-natural 2,5-diketopiperazine products will no longer be dependent on aminoacyl tRNA synthetase charging the unnatural amino acid onto the tRNA. Of course, synthesis of such non-natural 2,5-diketopiperazine products would require directed evolution of new CDPS.

#### We agree with the reviewer and have since emphasised and re-enforced our wording concerning the impact of the practicalities of using Leu-DBE as a substrate. This is presented in two different sentences added to the manuscript:
“Leu-DBE has a half-life of 431 mins in our assay conditions (Fig S20), and therefore Leu-DBE standards were run immediately after resuspension in water to reduce the hydrolysis associated with long term storage in water. ”
“The main requirement for successful protein design and in vitro evolution is sufficient measurable activity, allowing activity screens to be performed with enzyme variants, a condition fulfilled by Leu-DBE and BtCDPS.”

Furthermore, we synthesized Ile-DBE and Met-DBE, to demonstrate that the results obtained with misaminoacylated tRNAs could be replicated with the minimalistic DBE substrates, without the need to misaminoacylate tRNA substrates. This further illustrates the potential of these minimalistic substrates, both to understand substrate scope and for further development. These results were added to Fig. S11 – Alternative aa-DBE substrates.

- Section on Leu-PANS would need major changes.

#### Please see our above comment, which addresses this issue.

Minor concerns:
- I could not find the structure of S33A mutant in Figure 1, mentioned on page 3- “The crystal structure of full-length (residues 1-232) and S33A mutant enzymes was solved at a resolution of 1.69 Å and 1.78 Å, respectively (Fig. 1)”.

#### The structure of BtCDPSS33A was omitted from the manuscript due to the similarity to Wt BtCDPS structure. However, to address this concern we have added a supplementary figure (Fig.S13), which compares the structure of WT BtCDPS, BtCDPSS33A and BtCDPSR153A.

- Figure 1: It might be good to mention that the structure has 1,2-hexanediol (in pink).

#### We added the sentence to the figure legend “A 1,2-hexanediol bound to the substrate binding pocket of BtCDPS is shown in stick representation (pink).”.

- Figure 3C: “The amount of DBE-OH linearly increased with increasing enzyme concentration (Fig. 3C)”. Are there only three points that are used to make a straight line? The authors would need more data points.

#### We have rephrased this part on the manuscript to address this issue.
“The amount of DBE-OH formed was directly dependant on increasing enzyme concentration”

- It might be good to move Figure 3d after Figure 4.

#### We thank the reviewer’s suggestion, but think that Figure 3d (now Fig. 3F) should remain in its current order, accompanied by similar experiments involving our investigation into the Leu-DBE substrate.


Few other suggestions:
1. The suggestion that Arg153 might plays an important role in binding to the phosphate backbone of the tRNA was interesting. Given that Leu-DBE does not have the phosphate group. It might be good to test the activity of Arg153A (or L) mutants with Leu-DBE substrate.

#### Please see our comment above concerning R153A experiments. In summary we performed additional experiments with BtCDPSR153A, please see previous comment above.


2. It will be interesting to see if Leu-PANS can inhibit the activity of the enzyme.

#### Please see our comment above concerning Leu-PANS experiments.


Reviewer #3 (Remarks to the Author):

The authors report a piece of interesting work about Bacillus Thermoamylovorans cyclodipeptide synthases (BtCDPS), which can produce cyclic dipeptide products by coupling two aminoacylated tRNA substrates. The authors solved the structure of BtCDPS, explored its specificity toward different amino acid substrates, and particularly find a very simple start molecule, leucine dinitrobenzyl ester (Leu-DBE), instead of aa-tRNA as a substrate, which can largely facilitate the synthesis of cyclodipeptide. The conclusion are well supported by the experimental results. But I still have some concerns as below.

#### We thank the reviewer for their appraisal of our work

1) The kinetic curves of Leu-DBE cyclization catalyzed by BtCDPS and Leu-DBE hydrolysis are lacking.

#### To address this concern, we have since performed additional experiments monitoring BtCDPS activity throughout a time-period and performing quantification to determine an apparent rate. We added two panels to Figure 3 (Fig. 3D and E) to depict these time courses, and Supporting Figure 20 describes time courses for Leu-DBE hydrolysis (forming DBE-OH, monitored by HPLC) in the presence and absence of BtCDPS. We would like to point out that Leu-DBE hydrolyses in water, so performing kinetic measurements in which the concentration of Leu-DBE is varied are impractical as hydrolysis proceeds even after the reaction is quenched. In our materials and methods section, we highlight how we worked around the spontaneous Leu-DBE hydrolysis in water, to collect data points in the time course experiment.

2) The yield of Leu-DBE cyclization catalyzed by wt BtCDPS or even the S33C mutant is very low (1% or 2%). And the active amino ester is very easy to hydrolyze in the buffer. I am concerning about the improvement capacity of this enzyme.

#### There are several examples of enzymes that have been improved/evolved from very inefficient starting points. Several good examples can be found here2, in which total turnover numbers of 0 were converted into more than 500 after directed evolution.
Importantly, by analysing what a “moderately efficient enzyme”3 is we see naturally occurring enzymes that perform with kcat values at single digit h-1, and KM values in the triple digit mM range. Although inefficient catalysts, these proteins evolved and were selected to perform their function at low efficiency. When considering enzymes that were evolved to generate improved variants, these are typical ranges for starting points. Developing enzymes activities de novo has proven more challenging and produced less efficient catalysts than improving upon existing enzymatic activities.4 We therefore argue that the poor activity observed with Leu-DBE is a starting point for substrate and enzyme evolution, showing that the utilization of minimalistic substrates is possible. To illustrate this point, we added the sentence:

“The main requirement for successful protein design and in vitro evolution is sufficient measurable activity, allowing activity screens to be performed with enzyme variants, a condition fulfilled by Leu-DBE and BtCDPS. 4”

3) The authors mentioned BtCDPS is a thermostable enzyme. What is the activity of this enzyme toward different substrates at higher temperature?

#### The current assay conditions require tRNA synthetases which are not amenable to reaction at higher temperatures. Similarly, DBE-Leu stability decreases as temperature increases. The usefulness of thermophilic enzymes extends beyond reaction temperatures, however, since these proteins usually possess higher stability towards organics solvents5 and can offer simpler purification strategies, as well as posterior isolation and regeneration. An interesting perspective on cell-free chemical production highlights some of these qualities.6 We thank the reviewer for bringing this to our attention and added the following sentence to the manuscript:

“Thermophilic enzymes offer simpler strategies for protein isolation and regeneration6, and have been shown to possess higher tolerance to harsher reaction conditions, for example organic solvents.5”

1. L. Bonnefond, T. Arai, Y. Sakaguchi, T. Suzuki, R. Ishitani and O. Nureki, Proc Natl Acad Sci U S A, 2011, 108, 3912-3917.
2. R. Fasan, S. B. Jennifer Kan and H. Zhao, ACS Catal, 2019, 9, 9775-9788.
3. A. Bar-Even, R. Milo, E. Noor and D. S. Tawfik, Biochemistry, 2015, 54, 4969-4977.
4. C. Zeymer and D. Hilvert, Annu Rev Biochem, 2018, 87, 131-157.
5. J. Mansfeld and R. Ulbrich-Hofmann, Biotechnol Bioeng, 2007, 97, 672-679.
6. J. U. Bowie, S. Sherkhanov, T. P. Korman, M. A. Valliere, P. H. Opgenorth and H. Liu, Trends Biotechnol, 2020, 38, 766-778.

26-Dec-2020

Dear Dr Czekster:

Manuscript ID: CB-ART-08-2020-000142.R1
TITLE: Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg

Reviewer 3

The concerns were well addressed by the authors and I recommend to accept this manuscript as it is.

Reviewer 2

The authors have adequately addressed the concerns raised.

Minor edits:
1. Abstract: “setting the stage for the design of simpler catalytically improved substrates.” Not clear- It might be better to rephrase the sentence.
2. Pg. 5 “Moreover, this result also demonstrates BtCDPS is capable of using other tRNA bodies as long as the amino acid is also tolerated.” Not clear- It might be better to rephrase the sentence.
3. Label X-axis in Fig. 3C and 3F.