From the journal RSC Chemical Biology Peer review history

31-Jan-2020

Dear Professor van Aalten:

Manuscript ID: CB-ART-12-2019-000017
TITLE: A mechanism-inspired UDP-N-acetylglucosamine pyrophosphorylase inhibitor

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

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after minor revisions.

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Yours sincerely,

Cai-Guang Yang, Ph.D.
Associate Editor
RSC Chemical Biology

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Reviewer 1

The manuscript describes a detailed study of A. fumigatus UDP-N-acetylglucosamine pyrophosphorylase, containing crystal structures of the enzyme, study of active site residues using site-directed mutagenesis, and the synthesis and testing of a substrate analogue as a novel inhibitor. The study is detailed, and the structures will help to aid inhibitor design for this antifungal target in the future. I think the study will be of great interest in the field. I have some minor comments for the authors:

1. I found Figure 5 a bit confusing, for the UDPGlcNAc ligand it is hard to tell which phosphate is which. The authors could perhaps add labels for the uridine and GlcNAc termini.

2. It is a bit surprising that there are no protein ligands for the Mg2+ cofactor. Is that precedented? The authors do discuss the possibility of a second divalent metal ion site, that seems quite possible, given the amount of negative charge on the substrates.

3. The mechanism presented seems plausible. One point is that phosphotransfer reactions proceed via in-line displacements. It seems broadly in-line but hard to tell exactly from the figures what the bond angle is, perhaps the authors would like to comment on that.

Reviewer 2

UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyzes the biosynthesis of
uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by converting UTP and GlcNAc-1P to the sugar nucleotide. This enzyme is a potential antifungal target for inhibitor design. To probe the detailed mechanistic studies of UAP1, the authors determined three high resolution crystal structures of A. fumigatus UAP1, AfUAP1 complexed with GlcNAc-1P, AfUAP1 complexed with UTP, a Michaelis complex of AfUAP1 with UDP-GlcNAc, PPi and Mg2+. The structures not only unravel the key role the Mr2+ plays in coordinating the reactants/products but also show that binding of UDP-GlcNAc, magnesium and PPi to AfUAP-1 causes a considerable conformational change t of the protein. The authors further designed meUTP, an methylenebisphosphonate analogue of UTP as the inhibitor of AfUAP1, which inhibits enzyme activity with an IC50 of 115 ± 40 micromolar. The work is of great importance and written in clear and succinct way. It should be accepted after minor revision.

Suggestions
1. The authors should provide more details when they describe how to co-crystallize AfUAP1 with UTP, GlcNAc-1P and UDP-GlcNAc respectively.
2. It will be interesting to add more information of inhibitor assay, such as IC50 values towards different UAP1.
3. The font size in lines 22 and 23 of page 12 should be consistent with that in other parts.

Response to the referees’ comments


Reviewer 1

“I found Figure 5 a bit confusing, for the UDP-GlcNAc ligand it is hard to tell which phosphate is which. The authors could perhaps add labels for the uridine and GlcNAc termini”

Based on the reviewer’s suggestion, the UDP-GlcNAc has been labelled for the purpose of clarity.

“It is a bit surprising that there are no protein ligands for the Mg2+ cofactor. Is that precedented? The authors do discuss the possibility of a second divalent metal ion site, that seems quite possible, given the amount of negative charge on the substrates”

This is precedented because a similar structure from bacterial homologue (MtGlmU) revealed no interactions between the protein and Mg2+, but revealed the metal ion coordinating the highly negative substrates. For MtGlmU, a two divalent metal ions mechanism has been proposed; however, for AfUAP1 the position of the second metal is occupied by the positively charged residue K437, which we demonstrate to be important for catalysis.

“The mechanism presented seems plausible. One point is that phosphotransfer reactions proceed via in-line displacements. It seems broadly in-line but hard to tell exactly from the figures what the bond angle is, perhaps the authors would like to comment on that”

The bond angle is discussed in the text, 166o for AfUAP1 and 169o for MtGMU, in agreement with the in-line mechanism discussed by the referee. Additionally, and for better understanding, this has now been added in the figures and figure legend.


Reviewer 2

“The authors should provide more details when they describe how to co-crystallize AfUAP1 with UTP, GlcNAc-1P and UDP-GlcNAc respectively”

More details explaining the co-crystallization have been provided in the revised version of our manuscript (see the track changes in the methodology section).

“It will be interesting to add more information of inhibitor assay, such as IC50 values towards different UAP1”

We have tested the inhibitor on human UAP1 (also named as AGX1) and UAP1L1, and in both case we observed slight inhibition, but not sufficient to determine and report IC50s.

“The font size in lines 22 and 23 of page 12 should be consistent with that in other parts”

We observed that the font size of the lines mentioned by the reviewer is the same along the manuscript. However, we have ensured that the font size is uniform across the manuscript.

13-Feb-2020

Dear Professor van Aalten:

Manuscript ID: CB-ART-12-2019-000017.R1
TITLE: A mechanism-inspired UDP-N-acetylglucosamine pyrophosphorylase inhibitor

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 in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Professor Cai-Guang Yang
Associate Editor
RSC Chemical Biology

Reviewer 1

Thank you for the response, I am happy with the revised version.