From the journal RSC Chemical Biology Peer review history

13-Jun-2020

Dear Professor Süssmuth:

Manuscript ID: CB-REV-05-2020-000073
TITLE: Matters of Class: Coming of Age of Class III and IV Lanthipeptides

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.

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Associate Editor
RSC Chemical Biology

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

Reviewer 1

This review article focuses primarily on the biosynthesis of Class III and IV lanthipeptides, which are more rare and less well-studied than the class I and II peptides. The classification is made primarily based on the enzyme(s) that install the signature PTM of lanthipeptide. In class III and IV lanthipeptides, the enzyme is tripartite with domains corresponding to a lyase, a kinase, and a cyclase. Class III enzymes lack the Zn ions in the cyclase domain found in all other classes. Class III and IV lanthipeptides themselves also differ in structure and function. Many class III lanthipeptides include the labionin moiety while class IV lanthipeptides have a strongly conserved four ring pattern. The paper focuses primarily on the biosynthetic logic of these lanthipeptides with lengthy, detailed sections on 1) enzyme directionality, 2) proteolysis of the leader peptide, 3) function of each of the three domains with structural insights, 4) leader peptide binding, and 5) enzymes that further tailor lanthipeptides. The review is authoritative and well-referenced, and it will certainly be an asset to those who want to delve deeply into the enzymology of lanthipeptide biosynthesis. I have several suggestions that may make the paper accessible to broader readership below.

• The ordering of the sections was somewhat odd to me. The first two sections on directionality and proteolysis of the leader should appear after the sections on the enzymology and the role of the leader peptide, respectively
• Page 1, 2nd column, top: I would suggest providing a little detail about how class II dehydratases differ from the combo of class III/IV lyase/kinase. On the surface, both of them use ATP (or GTP for class III) to accomplish the same chemistry
• Page 1, 2nd column, middle: though SapB and AmfS were known for a while, their biosynthetic logic was not, right? It is worth clarifying when these were reclassified as Class III lanthipeptides
• Page 1, 2nd column, bottom: replace “is activating” with “activates” Likewise, page 2, column 1, bottom, replace “is catalyzing” with “catalyzes”
• Page 3, 1st column, top: the use of the word “topology” has become a little controversial in the RiPPs field because of its strict definition in mathematics and stereochemistry. See the recent paper on tryptorubin in Science by Clardy and Baran as an example. I would suggest removing all of the instances of “topology” and related terms and replacing them with more neutral terms like isomer, conformer, etc.
• Page 4, first column, middle: there is a section that states that information about whether the entire core peptide is dehydrated prior to cyclization is not known for class III and IV. Has this type of study been carried out for class I or II or is this a limitation for all lanthipeptides? This could be briefly addressed in the paper
• Page 4: this section about directionality and order in peptide processing is one of the only sections lacking a figure. It might help the more casual reader to include a figure that describes the key points of this section.
• Page 4, bottom of 2nd column: explain why the directionality is counterintuitive
• Page 5, first full paragraph: I’m not sure what microheterogenic means; should this be changed to heterogeneous?
• Page 5, 2nd column, the sentence about AlpA and AlpP talks about “aminopeptidase activity inherent to AlpP.” But my understanding is that AlpP cleaves at proline and AlpA provides that aminopeptidase activity. Does AlpP also have aminopeptidase activity?
• Page 8, 2nd paragraph in Lyase domain section: instead of just saying phosphate elimination, state that this enzyme results in Dhb formation
• Page 10, Fig 6 caption: it would be helpful to know which classes the three enzymes belong to
• Page 11, top of 2nd column: rephrase the sentence that says “…high homology is given when comparing…”. I think the authors meant to say “a given”
• Page 12, 2nd column: when describing the heterologous expression experiment in E. coli, another possibility is that the glycosyltransferase is sufficiently promiscuous that it can take an alternative sugar
• Page 15: ref 47 says “invalid citation”

Reviewer 2

This is a timely review focusing on the class III and class IV lanthipeptides. Lanthipeptides are among the best studied family of the RiPP superfamily. Compared to the class I and class II classes, members of the classes III and IV are relatively new, and the authors of this manuscript have done many important studies in the field. Overall, the manuscript is well organized and clearly written, and I really enjoyed reading it. I would support it is publication. A few minor issues should be fixed.

1. Page 1, “The precursors themselves can be subdivided into an Nterminal leader region, which is needed…” This sentence should be slightly modified because a few precursor peptides have N-terminal core and C-terminal follower peptide.
2. Page 1, “Class I uses a combination of discrete glutamyl-tRNAGludependent dehydratases.” According to the recent paper from van der Donk group (Ting et al. Science, 2019 365, 280), it is highly likely that many LanB enzymes use other aminoacyl-tRNA instead of glutamyl-tRNA. Consider changing glytamyl-tRNA to aminoacyl-tRNA.
3. Page 3“Without the usage of the sophisticated deuterium labeling strategy, the presence of such minor modification routes is hard to detect.” This sentence might be very true because MS-based methods can also be used to reveal complicate dehydration process, as shown by in PNAS paper by van der Donk and coworkers. Consider citing this paper (Zhang et al PNAS, 2014, 12031) because a MS-based sophisticated algorithm was used to show that the NisB-catalyzed dehydration is an overall N- to C-terminal process, but the directionality is not strict.
4. Same page “experiments with SbgL revealed a strict N-to-C-terminal directionality of the dehydration events in SgbA,9 which is similarly occurring for representatives of class I and II maturation enzymes.’’ Consider revising this sentence because, as pointed above, the dehydration catalyzed by the prototypic dehydrase NisB is not “strict” N-to-C.
5. Page 6 “FlaAcore” should be “FlaA core”
6. Page 12, “NRPS systems,68 have been known for a while now, the experimental validation of a mixed RiPP/PKS natural product enables more targeted discovery of novel RiPP hybrid systems”. Consider citing (Ting et al. Science, 2019 365, 280), which revealed a RiPP/NPRS hybrid system in natural product biosynthesis.
7. The drawing in Figure 4B, 5B, and 6B is not very clear, and this is particularly true for Figure 4B. I can only clearly see the residues and substrate by zooming in on my laptop, and apparently you cannot do this in a printed page. I would suggest removing the secondary structures in the zoom-in circle or increasing its the color brightness to clearly show the residues and the bonding substrates.

Prof. Dr. Cai-Guang Yang
Associate Editor RSC Chemical Biology
Shanghai Institute of Materia Medica
Chinese Academy of Sciences
555 Zu Chong Zhi Road
Zhang Jiang Hi-Tech Park
Pudong, Shanghai
P.R. China
Tel.: (+86) 021-50806029
June 25, 2020
Re: Resubmission of Manuscript CB-REV-05-2020-000073


Dear Prof. Yang,

Please find enclosed a copy of the revised manuscript “Matters of Class: Coming of Age of Class III and IV Lanthipeptides”. We were happy to see that both referees gave positive feedback and that they are in support of publication of this review article in RSC Chemical Biology. In response to the constructive criticism of the referees, the manuscript was revised accordingly.

To aid you in identifying the incorporated changes, an additional copy of the main text is uploaded in which these changes are highlighted in yellow. Below you will find point-by-point responses to the comments of the referees. I would also like to thank the referees for their critical evaluation that has improved the final manuscript.

Thank you for your consideration,

Julian D. Hegemann Roderich D. Süssmuth

Referee: 1
This review article focuses primarily on the biosynthesis of Class III and IV lanthipeptides, which are
more rare and less well-studied than the class I and II peptides. The classification is made primarily
based on the enzyme(s) that install the signature PTM of lanthipeptide. In class III and IV
lanthipeptides, the enzyme is tripartite with domains corresponding to a lyase, a kinase, and a
cyclase. Class III enzymes lack the Zn ions in the cyclase domain found in all other classes. Class III
and IV lanthipeptides themselves also differ in structure and function. Many class III lanthipeptides
include the labionin moiety while class IV lanthipeptides have a strongly conserved four ring pattern.
The paper focuses primarily on the biosynthetic logic of these lanthipeptides with lengthy, detailed
sections on 1) enzyme directionality, 2) proteolysis of the leader peptide, 3) function of each of the
three domains with structural insights, 4) leader peptide binding, and 5) enzymes that further tailor
lanthipeptides. The review is authoritative and well-referenced, and it will certainly be an asset to
those who want to delve deeply into the enzymology of lanthipeptide biosynthesis. I have several
suggestions that may make the paper accessible to broader readership below.
We thank the referee for these kind words and helpful comments to improve our manuscript. Each
comment is addressed below.
The ordering of the sections was somewhat odd to me. The first two sections on directionality and
proteolysis of the leader should appear after the sections on the enzymology and the role of the
leader peptide, respectively
We considered the suggested change, but in the end decided against incorporating it. The reason being
that we initially want to give the reader a more macroscopic overview of class III/IV lanthipeptide
biosynthesis by discussing the general directionality of the enzymatic processing and the removal of
the leader peptide before delving into the details of the catalytic mechanisms of each individual
domain and how they recognize their substrate. We think that thereby, the reader will already have a
firmer grasp of the overarching key elements of biosynthesis, before the biocatalytic principles are
addressed on a molecular level.
Page 1, 2nd column, top: I would suggest providing a little detail about how class II dehydratases
differ from the combo of class III/IV lyase/kinase. On the surface, both of them use ATP (or GTP for
class III) to accomplish the same chemistry
The revised manuscript now mentions the unique features of class II dehydratase domains in
comparison to class III/IV synthetases as suggested.
Page 1, 2nd column, middle: though SapB and AmfS were known for a while, their biosynthetic logic
was not, right? It is worth clarifying when these were reclassified as Class III lanthipeptides
We rephrased the according segment and now refer to the references where SapB and AmfS were first
considered as class III lanthipeptides.
Page 1, 2nd column, bottom: replace “is activating” with “activates” Likewise, page 2, column 1,
bottom, replace “is catalyzing” with “catalyzes”
These errors were corrected accordingly.
Page 3 of 28 RSC Chemical Biology
Page 3, 1st column, top: the use of the word “topology” has become a little controversial in the
RiPPs field because of its strict definition in mathematics and stereochemistry. See the recent paper
on tryptorubin in Science by Clardy and Baran as an example. I would suggest removing all of the
instances of “topology” and related terms and replacing them with more neutral terms like isomer,
conformer, etc.
We thank the referee to bring this controversy to our attention and replaced all instances of topology
as suggested.
Page 4, first column, middle: there is a section that states that information about whether the entire
core peptide is dehydrated prior to cyclization is not known for class III and IV. Has this type of study
been carried out for class I or II or is this a limitation for all lanthipeptides? This could be briefly
addressed in the paper
Indeed, several studies were carried out with class I and II systems on this topic. For class I systems, it
is easy to study the dehydration and the cyclization reactions separately as each process is carried out
by a distinct enzyme. Here, full dehydration can often be accomplished in absence of the cyclase.
However, dehydration reactions seem to happen more efficiently in the presence of the LanC protein
(even if the catalytic center of this LanC protein was inactivated through point mutations).
Furthermore, when both LanB and LanC enzymes are used in vitro at the same time, some cyclization
reactions are observed before full dehydration of the precursor is accomplished. Similar experiments
were performed after artificially separating the dehydratase and cyclase domains of class II proteins.
Again, it seems that the incorporation of the dehydrations can be independent of the cyclizations,
when compared in this manner. However, kinetic analysis of the processing reactions of several intact
class II enzymes revealed that some cyclization events almost always precede some of the dehydration
events. Indeed, these two processing steps and their coupling with each other appear to be rather
complex and the order of modification events might at least partially depend on the sequence and
modification state of the core peptide. In the revised manuscript we therefore now added a short
statement that in class I and II systems full dehydration can often be observed in absence of the cyclase,
but that in the presence of the cyclase usually some -thioether bridges are already installed before
the full dehydration of the core peptide is accomplished.
Page 4: this section about directionality and order in peptide processing is one of the only sections
lacking a figure. It might help the more casual reader to include a figure that describes the key points
of this section.
While the non-uniformity of observed processing routes for different compounds prevents us from
making a figure for generalization, we added a figure using the CurA processing by CurKC as an
example, as this is one of the most thoroughly studied systems in this regard and nicely demonstrates
a few exceptions when discussing overall directionality and processing. We also added a short
paragraph to the end of the segment highlighting that regarding processing directionality a
generalization is not possible and that there are at best some trends, which, however, could in part
have arisen from a sampling biases.
Page 4, bottom of 2nd column: explain why the directionality is counterintuitive
The sentence was rephrased for clarification.
Page 5, first full paragraph: I’m not sure what microheterogenic means; should this be changed to
heterogeneous?
The term microheterogenicity describes mixtures of compounds that share the same underlying
chemical scaffold but differ with regards to minor modifications of this scaffold like, e.g., different
patterns of methylations, hydroxylations, or in the case of lanthipeptides through different levels of
RSC Chemical Biology Page 4 of 28
Dha/Dhb and (Me)Lan incorporation or different remaining leader peptide residues still attached to
their N-terminus. It is used for example in context of natural product antibiotics that are isolated from
their native producers as such microheterogenic mixtures, e.g. peptaibol antibiotics.
Page 5, 2nd column, the sentence about AlpA and AlpP talks about “aminopeptidase activity
inherent to AlpP.” But my understanding is that AlpP cleaves at proline and AlpA provides that
aminopeptidase activity. Does AlpP also have aminopeptidase activity?
We modified the respective paragraph to make the statement clearer. In general, AlpP and homologs
were proposed to act as both endo- and aminopeptidases on their modified LanA substrates. In the
revised segment, we also added a short discussion about new findings on this matter from a recent
publication in Angewandte Chemie, where new AlpP homologs were characterized.
Page 8, 2nd paragraph in Lyase domain section: instead of just saying phosphate elimination, state
that this enzyme results in Dhb formation
This change was incorporated as suggested.
Page 10, Fig 6 caption: it would be helpful to know which classes the three enzymes belong to
We amended the figure caption as proposed.
Page 11, top of 2nd column: rephrase the sentence that says “…high homology is given when
comparing…”. I think the authors meant to say “a given”
This error was fixed in the revised version of the manuscript.
Page 12, 2nd column: when describing the heterologous expression experiment in E. coli, another
possibility is that the glycosyltransferase is sufficiently promiscuous that it can take an alternative
sugar
We agree with the referee that this is indeed a possibility and amended that sentence as proposed.
Page 15: ref 47 says “invalid citation”
We thank the referee for bringing this to our attention and fixed our citations accordingly!
Referee: 2
Comments to the Author
This is a timely review focusing on the class III and class IV lanthipeptides. Lanthipeptides are among
the best studied family of the RiPP superfamily. Compared to the class I and class II classes, members
of the classes III and IV are relatively new, and the authors of this manuscript have done many
important studies in the field. Overall, the manuscript is well organized and clearly written, and I
really enjoyed reading it. I would support it is publication. A few minor issues should be fixed.
We also would like to extend our gratitude for the helpful comments provided by referee #2. They will
be addressed in the following section.
Page 1, “The precursors themselves can be subdivided into an Nterminal leader region, which is
needed…” This sentence should be slightly modified because a few precursor peptides have Nterminal core and C-terminal follower peptide.
While some specific RiPPs certainly utilize C-terminal follower peptides for enzymatic recognition, the
N-terminal leader peptide is present in the vast majority of RiPPs, including all known lanthipeptides.
If we would elaborate on these rare exemptions, we would also need to discuss other examples outside
the lanthipeptide RiPP subfamily that slightly deviate from the general definition. For example, by
mentioning follower peptides in the introduction, we would also have to discuss the numbering
Page 5 of 28 RSC Chemical Biology
nomenclature in that light. However, we feel that this would not be for the benefit for a review article
focused on class III and IV lanthipeptides and might instead possibly be distracting, if not even
confusing, to some readers that are not that familiar with RiPPs. Hence, we decided against
incorporating this suggestion to have a more focused and concise review article.
Page 1, “Class I uses a combination of discrete glutamyl-tRNAGludependent dehydratases.”
According to the recent paper from van der Donk group (Ting et al. Science, 2019 365, 280), it is
highly likely that many LanB enzymes use other aminoacyl-tRNA instead of glutamyl-tRNA. Consider
changing glytamyl-tRNA to aminoacyl-tRNA.
As suggested, we changed “glutamyl-tRNA” to “aminoacyl-tRNA” and added the corresponding
citation.
Page 3“Without the usage of the sophisticated deuterium labeling strategy, the presence of such
minor modification routes is hard to detect.” This sentence might be very true because MS-based
methods can also be used to reveal complicate dehydration process, as shown by in PNAS paper by
van der Donk and coworkers. Consider citing this paper (Zhang et al PNAS, 2014, 12031) because a
MS-based sophisticated algorithm was used to show that the NisB-catalyzed dehydration is an
overall N- to C-terminal process, but the directionality is not strict.
We added this reference as proposed.
Same page “experiments with SbgL revealed a strict N-to-C-terminal directionality of the
dehydration events in SgbA,9 which is similarly occurring for representatives of class I and II
maturation enzymes.’’ Consider revising this sentence because, as pointed above, the dehydration
catalyzed by the prototypic dehydrase NisB is not “strict” N-to-C.
This is a good point. Considering that the experiments identifying the dehydration order of SgbL were
not done using sufficiently sophisticated methods that would have allowed the detection of minor
modification routes, like it was done for LabKC, we decided to change the “strict” to “preferred”, as,
like referee #2 pointed out, it is unlikely that such routes won’t exist in this case.
Page 6 “FlaAcore” should be “FlaA core”
This typo has been corrected.
The drawing in Figure 4B, 5B, and 6B is not very clear, and this is particularly true for Figure 4B. I can
only clearly see the residues and substrate by zooming in on my laptop, and apparently you cannot
do this in a printed page. I would suggest removing the secondary structures in the zoom-in circle or
increasing its the color brightness to clearly show the residues and the bonding substrates.
In order to increase the visibility of the catalytic residues and substrates in the zoom-in circles, we
made the secondary structures partially transparent, which highlights the relevant amino acid side
chains much more clearly. We did not altogether remove secondary structure motifs, as they are in
some cases important for biosynthesis, e.g. the P-loop and helix C for the kinase domains in Figure 4B
(5B in the revised manuscript).
Page 12, “NRPS systems,68 have been known for a while now, the experimental validation of a mixed
RiPP/PKS natural product enables more targeted discovery of novel RiPP hybrid systems”. Consider
citing (Ting et al. Science, 2019 365, 280), which revealed a RiPP/NPRS hybrid system in natural
product biosynthesis.
We have to strongly disagree with the referee on this matter. We think that referring to these peptideaminoacyl-tRNA-ligase(PEARL)-dependent natural products as non-ribosomal peptides or even
RiPP/NRPS hybrid systems is very misleading as traditional NRPS machineries have very little in
RSC Chemical Biology Page 6 of 28
common with the PEARL enzymes and as the resulting natural products are only technically
“synthesized non-ribosomally” (which is also true for many other natural product classes, like, e.g.,
cyclic dipeptidase synthases, whose biosynthesis is also aminoacyl-tRNA-dependent and which are not
considered to belong to the NRPS natural product superfamily. In fact, traditional NRPS machineries
also sometimes produce diketopiperazines, which are however strictly separated from CDPS-derived
compounds). Due to the lack of a ribosomally-synthesized core peptide for PEARL-dependent
biosynthesis, it could even be argued that these are a unique natural product superfamily instead of
an example for RiPPs. Thus, while we certainly agree that the secondary metabolites derived from
PEARL biocatalysis are highly interesting new natural products, we decided against mentioning them
in this article.
Additional changes:
Besides the changes requested by the referees, we changed two additional things in the revised
manuscript:
1) During the revision we found a minor mistake in the alignment of the lyase domains in Figure 5A.
We noticed that for SpvC and OspF the alignment algorithm introduces a gap of 5 residues before the
two catalytic Arg residues (R213 and R220 in SpvC). This alignment was similar to the one reported in
the original study of the VenL lyase domain (see Goto et al. Biochemistry 2011, 50(5), 891), hence we
assumed the alignment to be correct when preparing the original submission. However, we now
noticed that removal of this gap leads to a perfect alignment of SpvC R213/R220 with VenL R149/R156.
These two Arg residues are also conserved in almost all other class III and IV enzymes shown. We thus
performed a new alignment using only the last ~40 residues of each lyase domain and saw that the
gap disappears and all Arg residues align well:
SpvC GQCPESDVHPENWKYLSYRNELRSGRDGGEMQRQALRE
OspF GEYPASDVRPEDWKYVSYRNELRSDRNGSERQEQMLRE
StcL GPRILSDQPYAPQSLVHYRYGAFVGRRRVSEQGLLVWF
SgbL GPRILSDQPYAARSLVHYRYGAFVGRRRLSDDGLLVWF
VenL GPRILSDQPYAVNSLVHYRYGSFVGRRRLSDDGLLVWF
AciKC NPYILSDLRWNAGP-LHVRYGAFANRYTVSESGSVVPA
AplKC GPYILSDRRWRTGP-LFVRYGGFSPRHCRTATGELTPA
LabKC GPYILSDLRWRSGP-LFVRYGAFKEKFCRDGRGEMVPA
. ** : * :
Furthermore, we noticed that the Asp137 residue in VenL was indeed also conserved in OspF and SpvC.
As consequence, we extended our search for SpvC literature and indeed found that the corresponding
Asp201 in SpvC was observed to form a contact to the catalytic acid His106 and thereby is stabilizing
the protonated form of the imidazole group. Based on these new insights, we updated Figure 5 and
adjusted the corresponding segment of the paper accordingly.
2.) We also included short discussions and references to a new Angewandte Chemie publication that
was just released, which contains new insights into the lipolanthines and the endopeptidases involved
in the removal of their leader peptides. In this respect, we added updated and new information to the
segments discussing leader peptide removal, enzymatic recognition, and additional tailoring reactions.

08-Jul-2020

Dear Professor Süssmuth:

Manuscript ID: CB-REV-05-2020-000073.R1
TITLE: Matters of Class: Coming of Age of Class III and IV Lanthipeptides

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,

Dr Cai-Guang Yang
Associate Editor
RSC Chemical Biology

Reviewer 1

While I am still not convinced about the ordering of the sections, all of my other concerns have been addressed.