Reconstitution of Iterative Thioamidation in Closthioamide Biosynthesis Reveals Tailoring Strategy for Nonribosomal Peptide Backbones

Article abstract of DOI:10.1002/anie.201905992

Thioamide-containing nonribosomal peptides (NRPs) are exceedingly rare. Recently the biosynthetic gene cluster for the thioamidated NRP antibiotic closthioamide (CTA) was reported, however, the enzyme responsible for and the timing of thioamide formation remained enigmatic. Here, genome editing, biochemical assays, and mutational studies are used to demonstrate that an Fe-S cluster containing member of the adenine nucleotide α-hydrolase protein superfamily (CtaC) is responsible for sulfur incorporation during CTA biosynthesis. However, unlike all previously characterized members, CtaC functions in a thiotemplated manner. In addition to prompting a revision of the CTA biosynthetic pathway, the reconstitution of CtaC provides the first example of a NRP thioamide synthetase. Finally, CtaC is used as a bioinformatic handle to demonstrate that thioamidated NRP biosynthetic gene clusters are more widespread than previously appreciated.

Full text of DOI:10.1002/anie.201905992

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Accepted Article
Title: Reconstitution of Iterative Thioamidation in Closthioamide
Biosynthesis Reveals a Novel Nonribosomal Peptide Backbone-
Tailoring Strategy
Authors: Kyle L. Dunbar, Maria Dell, Evelyn M. Molloy, Florian Kloss,
and Christian Hertweck
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201905992
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10.1002/anie.201905992
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Reconstitution of Iterative Thioamidation in Closthioamide
Biosynthesis Reveals a Novel Nonribosomal Peptide Backbone-
Tailoring Strategy
Kyle L. Dunbar,[a] Maria Dell,[a] Evelyn M. Molloy,[a] Florian Kloss,[b] and Christian Hertweck*[a,c]
member of the adenine nucleotide α-hydrolase (AANH) protein
superfamily that processes thiotemplated substrates. Through
the use of substrate derivatives, we determine the timing of
thioamidation and revise the CTA biosynthetic pathway. Finally,
we leveraged the sequence of CtaC to demonstrate that diverse
anaerobic bacteria have the potential to produce thioamidated
NRPs.
Abstract: Thioamide-containing nonribosomal peptides (NRPs) are
exceedingly rare. Recently the biosynthetic gene cluster for the
thioamidated NRP antibiotic closthioamide (CTA) was reported;
however, the enzyme responsible for and the timing of thioamide
formation remained enigmatic. Here, we use genome editing,
biochemical assays and mutational studies to demonstrate that an
Fe-S cluster-containing member of the adenine nucleotide α-
hydrolase protein superfamily (CtaC) is responsible for sulfur
incorporation during CTA biosynthesis. However, unlike all
Of the genes present in the CTA biosynthetic gene cluster,
we predicted that the product of ctaC (Ccel_3258) would be
responsible for thioamide biosynthesis as it is a member of the
AANH protein superfamily (Figure 1C and Supplementary
Information Figure S1).[5] Members of this superfamily catalyze
transformations ranging from cofactor and antibiotic biosynthesis
to the posttranslational modification of proteins.[7] Although these
reactions are varied, all involve ATP-dependent adenylation of
the substrate followed by nucleophilic attack at the activated
position and elimination of AMP. Notably, some AANH proteins
perform oxygen-by-sulfur substitutions on nucleotides in both
primary and secondary metabolic pathways.[6b, 7-8] However, no
AANH protein is known to thioamidate a peptidic substrate.
While analysis of the metabolite profile of CtaC-deficient R.
cellulolyticum indicated that CtaC is essential for CTA
biosynthesis, no biosynthetic intermediates could be detected to
clarify the role of this uncharacterized AANH protein.[5] However,
it is possible that the type II intron inserted in ctaC causes polar
effects.[9] We therefore used a CRISPR-Cas9 genome editing
method to generate a new R. cellulolyticum ΔctaC strain with a
nonsense mutation in ctaC (Figure S2);[5, 10] however, no oxygen
isosteres of known CTA congeners were detected in culture
extracts (Figure S3). We next turned to an Escherichia coli
heterologous expression system for the production of
polythioamidated peptides.[5] A vector containing all of the CTA
biosynthetic genes except ctaC was constructed (pCTA-ΔctaC;
Figure S4) and the metabolite profile of E. coli pCTA-ΔctaC was
checked for the accumulation of intermediates. Consistent with
the R. cellulolyticum ΔctaC results, no thioamide-free
intermediates were detected (Figure S4).
previously characterized members, CtaC functions in
a
thiotemplated manner. In addition to prompting a revision of the CTA
biosynthetic pathway, the reconstitution of CtaC provides the first
example of a NRP thioamide synthetase. Finally, using CtaC as a
bioinformatic handle, we provide promising clues that thioamidated
NRP biosynthetic gene clusters are more widespread than
previously appreciated.
Thioamides are formed through the oxygen-by-sulfur substitution
of amide bonds, a seemingly minor change that has major
repercussions for the physicochemical properties of the
compound.[1] Accordingly, site-specific thioamidation has long
been used by synthetic chemists to tune the properties of small
molecules. In regard to natural products, only a modest number
2]
of thioamidated secondary metabolites are known.[1c,
One
notable example is closthioamide (CTA, Figure 1A), a symmetric
nonribosomal peptide (NRP) produced by the anaerobic
bacterium Ruminiclostridium cellulolyticum DSM 5812.[3] This
DNA gyrase inhibitor harbors six thioamide linkages that are
essential for its antimicrobial activity and is the only bacterial
4]
thioamide-containing
NRP
identified
to
date.[3a,
Recently, we demonstrated that CTA is biosynthesized by
an unusual thiotemplated, NRP synthetase (NRPS)-independent
pathway (Figure 1B) and proposed
a model for CTA
maturation.[5] However, the enzyme responsible for thioamide
biosynthesis and the timing of thioamide formation remained
unknown. Indeed, nothing is known about how these moieties
are made in NRPs, in contrast to the great strides made towards
In order to circumvent the difficulties in defining the role of
CtaC by genetic approaches, we set out to characterize CtaC in
vitro. Owing to solubility issues with His6-tagged CtaC, the
protein was isolated as an N-terminal maltose binding protein
(MBP)-tagged fusion protein (Figure S5). During isolation we
noted that aerobically purified MBP-CtaC solutions were slightly
red, potentially indicating that CtaC binds iron. A recently
identified tRNA-modifying subfamily (TtcA/TtuA/Ncs6) of the
AANH superfamily was shown to require an iron-sulfur cluster
for activity.[11] Indeed, CtaC has the [4Fe-4S] cluster-binding
motif found in TtcA/TtuA proteins (Figure S6).[11a, 11c, 11d] However,
understanding thioamide formation in both ribosomal peptides
6]
and nucleoside antimetabolites.[1c,
Here, we identify and
reconstitute the activity of a NRP thioamide synthetase (CtaC)
involved in CTA maturation. Through phylogenetic analyses and
biochemical assays, we demonstrate that CtaC is a novel
[a]
Dr. K. L. Dunbar, M. Dell, Dr. E. M. Molloy, Prof. Dr. C. Hertweck
Dept. of Biomolecular Chemistry, Leibniz Institute for Natural
Product Research and Infection Biology, HKI
Beutenbergstrasse 11a, 07745 Jena (Germany)
E-mail: christian.hertweck@leibniz-hki.de
Dr. F. Kloss
Transfer Group Antiinfectives, Leibniz Institute for Natural Product
Research and Infection Biology, HKI
Beutenbergstrasse 11a, 07745 Jena (Germany)
Prof. Dr. C. Hertweck
[b]
[c]
the UV-visible spectrum of MBP-CtaC had only
a weak
absorbance at the characteristic wavelength for such metal
centers (~410 nm,[11a, 11c, 11d] Figure 2A) and purified with just 1.2
± 0.1 iron and 0.9 ± 0.1 sulfide bound per protomer. To obtain
MBP-CtaC with an appropriately assembled iron-sulfur cluster,
the protein was treated with sulfide and ferrous iron in an
anaerobic glovebox. The resultant MBP-CtaC solution adopted a
brownish-red color, had an enhanced absorbance at 410 nm
Chair of Natural Product Chemistry
Friedrich Schiller University Jena, 07743 (Germany)
Supporting information for this article is given via a link at the end of
the document.
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were performed in an anaerobic glovebox and progress was
monitored by MALDI-TOF-MS. Although no processing of 1-
holo-CtaH (Figure S8) or 2-holo-CtaE was observed (Figure S9),
the mass of 1-holo-CtaE increased by approximately 32 Da in
reactions compared to controls (Figure 2C). This shift is
consistent with the installation of two thioamides and was
dependent on the presence of CtaC, ATP and sulfide. To
definitively identify the product of the CtaC reaction, the thioester
linking 1 to holo-CtaE was hydrolyzed, and the released
products were analyzed by LC-HR-MS. A peak with a retention
time and mass consistent with di-thioamidated 1 was only
observed in reactions containing all components (Figure 2D,
Figure S10). Furthermore, MS/MS analysis of the product
localized the sulfur atoms to the amide bonds (Figure S10).
In order to probe the substrate scope of CtaC,
thioamidation reactions were performed on substrates not linked
to CtaE (1, 1-CoA, 3-CoA, 4, and 5; Figure 2B). Despite using
elevated enzyme concentrations and long reaction times, no
turnover was observed by LC-HR-MS (Figure S11–S15).
Together, these data demonstrate that CtaC is a thiotemplated
thioamide synthetase that functions on CtaE-bound substrates
and that sulfur insertion occurs after HBA is added to the poly-β-
alanine backbone.
AANH enzymes adenylate their substrates and thus
produce AMP as a byproduct of the reaction.[7] When we
measured the ATP hydrolysis products from thioamidation
reactions with 1-holo-CtaE by HPLC, both AMP and ADP were
detected (Figure S16). Moreover, neither the production of AMP
nor ADP correlated with substrate processing by CtaC (Figure
S16). Although members of the AANH superfamily are highly
divergent in both primary sequence and function, they share a
fully conserved ATP-binding motif that is used to present ATP
for substrate adenylation.[7] Thus, as an alternative approach to
investigate the ATP-dependent activity of CtaC, we generated
mutant proteins bearing single alanine substitutions in the ATP-
binding pocket (CtaCD77A and CtaCS78A, Figure S5–S6) and
assayed the mutants for their ability to thioamidate 1-holo-CtaE.
As predicted, the activity of both mutants was abrogated (Figure
2E, Figure S17).
Figure 1. The CTA gene cluster contains an AANH homolog. (A) Structure
of CTA. (B) The CTA biosynthetic gene cluster. AANH, adenine nucleotide α-
hydrolase; PCP, peptidyl carrier protein. (C) Neighbor-joining phylogenetic tree
of AANH proteins (1000 bootstrap replicates). Structures of the compounds
biosynthesized by each AANH subclass are shown with the installed moiety
colored red. See Supporting Information for a fully annotated cladogram.
(Figure 2A), and contained 4.5 ± 0.2 iron and 4.5 ± 0.1 sulfide
per protomer.
Based on the thiotemplated nature of CTA biosynthesis,
we previously hypothesized that thioamide formation would
occur on substrates linked to one of the two peptidyl carrier
proteins (PCPs; CtaE/CtaH) encoded in the biosynthetic gene
cluster (Figure 1B).[5] Thus, we purified CtaE and CtaH as N-
terminal His6-tagged fusions and used matrix-assisted laser
desorption/ionization-time-of-flight-mass spectrometry (MALDI-
TOF-MS) to verify that they were in their apo form (i.e. lacking
the phosphopantetheinyl arm, Figure S7). As the timing of
thioamide formation during CTA maturation was unknown, we
synthesized coenzyme A (CoA)-conjugates differing in the
presence or absence of 4-hydroxybenzoic acid (HBA) (1-CoA
and 2-CoA; Figure 2B). These synthetic CoA-linked substrates
were loaded on the apo-PCPs by the promiscuous
phosphopantetheinyl transferase (PPTase) Sfp to generate the
corresponding peptidyl-holo-PCPs (e.g. 1-holo-CtaE; Figure
S7).[12]
As mentioned above, members of the TtcA/TtuA family of
sulfur-inserting AANH enzymes require an oxygen-labile [4Fe-
4S] cluster coordinated by three conserved cysteine residues for
11c, 11d]
activity.[11a,
Although the precise role is unclear, the
prevailing hypothesis is that the cluster coordinates sulfide at the
unique iron site and activates it for insertion into the substrate.[11]
To investigate the role of the Fe-S cluster in thioamide formation
by CtaC, we generated mutant proteins bearing single alanine
substitutions to residues predicted to be involved in Fe-S cluster-
binding (CtaCC154A, CtaCC157A, and CtaCC284A, Figure S5–S6).
While all mutant proteins bound similar levels of iron and sulfide
as wild-type CtaC (Figure S18), their thioamidation activity was
diminished (Figure 2E, Figure S17). These results establish that
although single mutations to the Fe-S cluster binding site are not
sufficient to fully disrupt cluster coordination, the activity of CtaC
is highly sensitive to perturbations to its metal binding site.
Notably, this phenomenon has been observed for other Fe-S
cluster dependent AANH enzymes.[11a, 11d] In further support of
the essential nature of the metal center for CtaC activity,
processing of 1-holo-CtaE was completely lost in thioamidation
reactions performed with CtaC under aerobic conditions or with
CtaC stripped of the Fe-S cluster (Figure 2E, Figure S17).
Over the course of our studies we noted that regardless of
the reaction time and concentration of CtaC used, tri-
With appropriately loaded PCPs prepared, we tested CtaC
for thioamidation activity. As all AANH superfamily members
require ATP for activity,[7] thioamidation reactions were
supplemented with ATP and magnesium. Lithium sulfide acted
as the sulfur donor since a sulfur-mobilization enzyme is not
encoded in the CTA biosynthetic gene cluster and other sulfur-
inserting AANH enzymes have been shown to accept this non-
13]
native sulfur donor in vitro.[11b-d,
All thioamidation reactions
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Figure 2. CtaC is a PCP-dependent thioamide synthetase. (A) UV-visible absorbance spectra of as-isolated and reconstituted MBP-CtaC. (B) Structures of
substrates used in this study. (C) Representative MALDI-TOF-MS spectral overlay of 6 h in vitro thioamidation reactions with 1-holo-CtaE. (D) LC-HR-MS profiles
of thioamidation reactions with 1-holo-CtaE following thioester cleavage. Traces correspond to the extracted ion chromatogram of the [M-H]- ionic species for di-
thioamidated 1 and are displayed with m/z values ± 5 ppm from the calculated exact mass. (E) Representative MALDI-TOF-MS spectral overlay of 22 h in vitro
thioamidation reactions with 1-holo-CtaE and mutant CtaC enzymes, CtaC under aerobic conditions (+ O2), or apo-CtaC (– Fe-S). (F) Representative MALDI-
TOF-MS spectral overlay of 22 h thioamidation reactions with 3-holo-CtaE. (G) Updated biosynthetic scheme for CTA maturation. (C, E, F) Guide lines denote the
expected shifts (+ 16 Da) obtained from the substitution of oxygen by sulfur. Red line: heat-inactivated CtaC.
thioamidated 1-holo-CtaE was never detected (Figure S19),
accounting for only four of the six thioamides present in CTA.
The initial biosynthetic model for CTA maturation posited that
CtaC installs the final two thioamides after the dimerization of di-
thioamidated 1 through diaminopropane (DAP);[5] however, the
inability of CtaC to process free substrates casts doubt on this
proposal. Indeed, products bearing three or four thioamidated β-
alanine residues were detected from R. cellulolyticum and the E.
2F). Furthermore, using a combination of LC-HR-MS and LC-
HR-MS/MS, the product was assigned as tri-thioamidated 3
(Figure S20).
In light of these data, we propose a revised biosynthetic
pathway for CTA maturation (Figure 2G). Briefly, the ATP-grasp
(CtaD) and acetyl-CoA synthetase (CtaI) proteins are predicted
to load β-alanine and HBA onto CtaE and CtaH, respectively.
HBA is synthesized from chorismate by CtaA[5] and β-alanine is
either biosynthesized from aspartate by the decarboxylase CtaF
or taken from primary metabolism. The peptidyl transferase
CtaG would transfer HBA from CtaH to tri-β-alanine-CtaE to
form 3-CtaE. Following the thioamidation of 3-CtaE by CtaC,
DAP — which is potentially synthesized from aspartate by the
coli heterologous expression system, although their biosynthetic
5]
relevance was unknown.[3b,
We therefore reasoned that a
CtaE-bound substrate containing three, rather than two, β-
alanine residues is the on-pathway substrate for thioamidation
by CtaC. Accordingly, 3-CoA (Figure 2B) was synthesized,
loaded onto CtaE in vitro using Sfp, and the conjugate (3-holo-
CtaE) was tested for processing by CtaC. The MALDI-TOF-MS
spectra demonstrated that the mass of 3-holo-CtaE increased by
approximately 48 Da in reactions compared to controls (Figure
sequential action of
a reductase (CtaK), aminotransferase
(CtaB), and decarboxylase (CtaF) — would be coupled to two
molecules of tri-thioamidated 3 by the transglutaminase CtaJ to
form CTA.[5] We suggest that the terminal β-alanine remains on
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found in anaerobic bacteria (Table S5), suggesting that
anaerobes represent an untapped trove of thioamidated NRPs.
In summary, we have reconstituted the first instance of
thioamide formation in a NRP. The data presented herein
demonstrate that this unusual thioamide synthetase functions on
thiotemplated substrates and represents a new subfamily of the
Fe-S cluster dependent AANH enzymes. In addition to further
clarifying the CTA biosynthetic pathway, the characterization
CtaC serves as a foundation for the discovery of additional
thioamidated NRPs. Future studies will focus on unearthing the
native sulfur source for this enzyme and the characterization of
the remaining steps in CTA maturation.
Acknowledgements
We thank A. Perner, T. Kindel and M. García-Altares Peréz for
MS measurements and H. Heinecke for NMR measurements.
We are grateful to K. Ishida and J. Kumpfmüller for technical
advice. K.L.D. and F.K. were supported by the Humboldt
Research Fellowship for Postdoctoral Researchers and
InfectControl 2020 (FKZ 03ZZ0803A), respectively. Financial
support by the DFG (Leibniz Award to C.H.) is gratefully
acknowledged.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: antibiotics • biosynthesis • enzymes • natural
products • thioamide
[1] a) A. Choudhary, R. T. Raines, Chembiochem 2011, 12, 1801-1807; b)
E. J. Petersson, J. M. Goldberg, R. F. Wissner, Phys. Chem. Chem.
Phys. 2014, 16, 6827-6837; c) N. Mahanta, D. M. Szantai-Kis, E. J.
Petersson, D. A. Mitchell, ACS Chem. Biol. 2019, 14, 142-163.
[2] a) K. L. Dunbar, D. H. Scharf, A. Litomska, C. Hertweck, Chem. Rev.
2017, 117, 5521-5577; b) S. Banala, R. D. Sussmuth, Chembiochem
2010, 11, 1335-1337.
[3] a) T. Lincke, S. Behnken, K. Ishida, M. Roth, C. Hertweck, Angew.
Chem. Int. Ed. 2010, 49, 2011-2013; b) S. Behnken, T. Lincke, F. Kloss,
K. Ishida, C. Hertweck, Angew. Chem. Int. Ed. 2012, 51, 2425-2428.
[4] a) A. I. Chiriac, F. Kloss, J. Kramer, C. Vuong, C. Hertweck, H. G. Sahl,
J. Antimicrob. Chemother. 2015, 70, 2576-2588; b) V. F. Miari, P.
Solanki, Y. Hleba, R. A. Stabler, J. T. Heap, Antimicrob. Agents
Chemother. 2017, 61; c) F. Kloss, A. I. Chiriac, C. Hertweck, Chem. Eur.
J. 2014, 20, 15451-15458.
[5] K. L. Dunbar, H. Buttner, E. M. Molloy, M. Dell, J. Kumpfmuller, C.
Hertweck, Angew. Chem. Int. Ed. 2018, 57, 14080-14084.
[6] a) N. Mahanta, A. Liu, S. Dong, S. K. Nair, D. A. Mitchell, Proc. Natl.
Acad. Sci. U.S.A. 2018, 115, 3030-3035; b) A. Litomska, K. Ishida, K. L.
Dunbar, M. Boettger, S. Coyne, C. Hertweck, Angew. Chem. Int. Ed.
2018, 57, 11574-11578; c) G. E. Kenney, L. M. K. Dassama, M. E.
Pandelia, A. S. Gizzi, R. J. Martinie, P. Gao, C. J. DeHart, L. F.
Schachner, O. S. Skinner, S. Y. Ro, X. Zhu, M. Sadek, P. M. Thomas, S.
C. Almo, J. M. Bollinger, Jr., C. Krebs, N. L. Kelleher, A. C. Rosenzweig,
Science 2018, 359, 1411-1416; d) D. D. Nayak, N. Mahanta, D. A.
Mitchell, W. W. Metcalf, Elife 2017, 6; e) J. Liu, Z. Lin, Y. Li, Q. Zheng, D.
Chen, W. Liu, Org. Biomol. Chem. 2019, 17, 3727-3731; f) C. J.
Schwalen, G. A. Hudson, B. Kille, D. A. Mitchell, J. Am. Chem. Soc.
2018, 140, 9494-9501.
Figure 3. Homologs of CtaC are found in diverse thiotemplated
biosynthetic pathways. (A) Sequence similarity network of CtaC homologs.
Nodes are colored based on the class of biosynthetic machinery found near
the CtaC homolog, while their shape denotes the phylum of the organism
harboring the gene cluster. NCT, non-canonical thiotemplate; PKS, polyketide
synthase. (B) Representative biosynthetic gene clusters that encode a CtaC
homolog (selected based on architecture diversity). Numbers correspond to
the node numbering in panel A. rSAM, radical S-adenosylmethionine enzyme.
CtaE, thus serving as a priming residue that allows CtaC to
concurrently install all thioamides in chemically equivalent
positions (Figure 2G).
As CtaC is the first example of
a NRP thioamide
synthetase, we sought to determine if this strategy for
thioamidated NRP biosynthesis exists outside CTA maturation.
To this end, we retrieved 100 homologs of CtaC from GenBank
and generated a sequence similarity network with the protein
sequences (Figure 3A).[14] Next, we surveyed the genomic
neighborhood surrounding the ctaC homolog for genes encoding
secondary metabolite biosynthetic enzymes (Figure 3A). Notably,
genes encoding thiotemplated biosynthetic enzymes were found
in approximately 75% of cases. In addition to a small number of
CTA-like biosynthetic gene clusters, many CtaC homologs are
encoded in the vicinity of NRPSs (Figure 3B), suggesting that
this strategy for thioamidation is also used by canonical NRP
biosynthetic pathways. Moreover, the majority of homologs were
[7] M. Fellner, R. P. Hausinger, J. Hu, Crit. Rev. Biochem. Mol. Biol. 2018,
53, 607-622.
[8] a) N. Shigi, Front. Microbiol. 2018, 9, 2679; b) S. Coyne, C. Chizzali, M.
N. Khalil, A. Litomska, K. Richter, L. Beerhues, C. Hertweck, Angew.
Chem. Int. Ed. 2013, 52, 10564-10568.
[9] K. N. McAllister, L. Bouillaut, J. N. Kahn, W. T. Self, J. A. Sorg, Sci. Rep.
2017, 7, 14672.
[10] T. Xu, Y. Li, Z. Shi, C. L. Hemme, Y. Li, Y. Zhu, J. D. Van Nostrand, Z.
He, J. Zhou, Appl. Environ. Microbiol. 2015, 81, 4423-4431.
[11] a) D. Bouvier, N. Labessan, M. Clemancey, J. M. Latour, J. L. Ravanat,
M. Fontecave, M. Atta, Nucleic Acids Res. 2014, 42, 7960-7970; b) Y.
Liu, D. J. Vinyard, M. E. Reesbeck, T. Suzuki, K. Manakongtreecheep, P.
This article is protected by copyright. All rights reserved.

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L. Holland, G. W. Brudvig, D. Soll, Proc. Natl. Acad. Sci. U.S.A. 2016,
113, 12703-12708; c) S. Arragain, O. Bimai, P. Legrand, S. Caillat, J. L.
Ravanat, N. Touati, L. Binet, M. Atta, M. Fontecave, B. Golinelli-
Pimpaneau, Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 7355-7360; d) M.
Chen, S. I. Asai, S. Narai, S. Nambu, N. Omura, Y. Sakaguchi, T. Suzuki,
M. Ikeda-Saito, K. Watanabe, M. Yao, N. Shigi, Y. Tanaka, Proc. Natl.
Acad. Sci. U.S.A. 2017, 114, 4954-4959.
[12] L. E. Quadri, P. H. Weinreb, M. Lei, M. M. Nakano, P. Zuber, C. T.
Walsh, Biochemistry 1998, 37, 1585-1595.
[13] C. T. Lauhon, W. M. Erwin, G. N. Ton, J. Biol. Chem. 2004, 279, 23022-
23029.
[14] J. A. Gerlt, J. T. Bouvier, D. B. Davidson, H. J. Imker, B. Sadkhin, D. R.
Slater, K. L. Whalen, Biochim. Biophys. Acta 2015, 1854, 1019-1037.
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Entry for Table of Contents:
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K.L. Dunbar, M. Dell, E. M. Molloy, F.
Kloss, C. Hertweck*
Page No. – Page No.
Reconstitution of Iterative
Thioamidation in Closthioamide
Biosynthesis Reveals a Novel
Nonribosomal Peptide Backbone-
Tailoring Strategy
Thiotemplated thioamidation: Closthioamide (CTA) is a thioamide-containing
nonribosomal peptide (NRP). The thioamide synthetase responsible for sulfur
insertion in CTA assembly was identified and characterized in vitro, shedding new
light on the thiotemplated assembly of this unusual antibiotic. Bioinformatic
analyses indicate that anaerobic bacteria may represent an untapped source for
thioamidated NRPs.
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