Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor

Article abstract of DOI:10.1002/anie.202003958

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

Full text of DOI:10.1002/anie.202003958

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Angewandte
Chemie
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Accepted Article
Title: Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly
Line Construct the Cyclopropanol Warhead of a Burkholderia
Virulence Factor
Authors: Felix Trottmann, Keishi Ishida, Jakob Franke, Aleksa Stanišić,
Mie Ishida-Ito, Hajo Kries, Georg Pohnert, and Christian
Hertweck
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202003958
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Angewandte Chemie International Edition
Biosynthesis
DOI: 10.1002/anie.200((will be filled in by the editorial staff))
Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line
Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor
Felix Trottmann, Keishi Ishida, Jakob Franke, Aleksa Stanišić, Mie Ishida-Ito, Hajo Kries, Georg Pohnert,
and Christian Hertweck*
Abstract: Pathogenic bacteria of the Burkholderia pseudomallei
group cause severe infectious diseases such as glanders and
melioidosis. Malleicyprols were identified as important bacterial
virulence factors, yet the biosynthetic origin of their cyclopropanol
warhead has remained enigmatic. By a combination of mutational
analysis and metabolomics we found that sulfonium acids,
dimethylsulfoniumpropionate (DMSP) and gonyol, known as
warfare agents[2] and a threat to global health.[3] Since infections
by these notorious pathogens are difficult to treat,[4] novel
therapeutic approaches such as anti-virulence strategies are
needed. As prerequisite to disarming pathogens, it is essential to
understand their virulence factors and the biosynthetic pathways
involved.[5] For B. pseudomallei and related pathogens such as
the less virulent model organism B. thailandensis, various
osmolytes and as crucial components in the global organosulfur cycle, macromolecular[6] and low-molecular-weight virulence factors[3, 7]
are key intermediates en route to the cyclopropanol unit. Functional
genetics and in vitro analyses uncover a specialized pathway to
DMSP involving a rare prokaryotic SET-domain methyltransferase for
a cryptic methylation, and show that DMSP is loaded onto the NRPS-
PKS hybrid assembly line by an adenylation domain dedicated to
zwitterionic starter units. Then, the megasynthase transforms DMSP
into gonyol, as demonstrated by heterologous pathway reconstitution
in E. coli.
have been identified. Notably, all pathogens of the B.
pseudomallei complex share a gene locus coding for an unusual
modular thiotemplate assembly line with components of modular
non-ribosomal peptide synthetases (NRPS) and polyketide
synthases (PKS). Gene inactivation experiments unequivocally
linked this gene cluster, named bur, to pathogenicity in
nematode[8] and mouse models.[9] However, the first metabolite
associated to this pathway
-
burkholderic acid[10] syn.
malleilactone[8] (1) - did not exhibit any activity explaining the
phenotypes observed in the infection models.
Burkholderia pseudomallei group pathogens cause lethal
infections that are difficult to treat.[1] The best-known members of
this pathogen complex are Burkholderia mallei, which causes the
zoonotic disease glanders, and B. pseudomallei, the causative
agent of melioidosis. The low infective dose needed and the
possibility of infections through inhalation have led to the
classification of B. mallei and B. pseudomallei as biological
Recently, we found 1 to be the inactivated form of the true
virulence factor,
a highly reactive, cyclopropanol-substituted
congener named bis-malleicyprol (2a, Figure 1A) formed from the
monomer malleicyprol (2b).[11] Nematode and toxicity assays
showed dramatically increased activity of 2a compared to 1,
implicating the cyclopropanol warhead in virulence.[11] Thus,
understanding its biosynthesis would set the basis for
antivirulence strategies. According to stable-isotope labeling
experiments and gene knockouts, the NRPS-PKS hybrid enzyme
BurA assembles the cyclopropanol-containing fragment of 2b,
followed by dimerization to 2a, which opens to form the inactive
propanone-substituted unit of 1, from a yet unknown methionine
(3)-derived C3 building block and malonyl-CoA (Figure 1B).[10] Yet,
structures and biotransformations of the precursors loaded onto
BurA have remained a riddle. Here we decipher the biogenetic
origin of the rare cyclopropanol warhead of malleicyprol and show
that a set of zwitterionic sulfonium acids initiates biosynthesis that
play key roles in global sulfur cycling.
[∗]
F. Trottmann, Dr. K. Ishida, Dr. M. Ishida-Ito Prof. Dr. C. Hertweck
Department of Biomolecular Chemistry, Leibniz Institute for Natural
Product Research and Infection Biology (HKI)
Beutenbergstr. 11a, 07745 Jena, Germany
Fax: (+) 49 3641 5320804
E-mail: christian.hertweck@leibniz-hki.de
Homepage: www.leibniz-hki.de
A. Stanišić, Dr. H. Kries
Junior Research Group Biosynthethic Design of Natural Products,
Leibniz Institute for Natural Product Research and Infection Biology
(HKI)
Beutenbergstr. 11a, 07745 Jena, Germany
Prof. Dr. J. Franke
Institute of Botany,
Leibniz University Hannover, 30419 Hannover, Germany
Prof. Dr. G. Pohnert
Institute for Inorganic and Analytical Chemistry, Friedrich Schiller
University Jena, 07743 Jena, Germany
Prof. Dr. C. Hertweck
Natural Product Chemistry, Faculty of Biological Sciences,
Friedrich Schiller University Jena, 07743 Jena, Germany.
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under: https://doi.org/####
This article is protected by copyright. All rights reserved.

10.1002/anie.202003958
Angewandte Chemie International Edition
Figure 1. A) Structures of burkholderic acid (1) and bis-malleicyprol (2a)
featuring
a
cyclopropanol warhead. B) Isotope-labeling studies[10] suggest
acetate and methionine (3) as precursors to malleicyprol (2b), the monomer[11]
of 2a.
To elucidate the biogenetic origin of the malleicyprol
warhead we first focused on the NRPS-PKS hybrid enzyme BurA
and its unknown starter unit. Specifically, we tested for
accumulation of intermediates in a mutant of the malleicyprol
overexpressing strain B. thailandensis Pbur lacking a functional
copy of burA (B. thailandensis PburΔburA[10]). Candidates for the
sulfur-containing pathway intermediates, however, could not be
detected by routine HR-LCMS-based metabolic profiling. Only a
comparative metabolomics analysis (Pbur vs. PburΔburA)
searching for highly polar metabolites revealed the elusive
methionine-derived starter unit. Compound 4 with m/z 135
accumulates in B. thailandensis PburΔburA cells (Figure 2A).
Based on its exact mass (m/z 135.0474; [M+H]+) we deduced the
molecular formula (C5H11O2S) of 4. By comparison with authentic
Figure 2. Identification of sulfonium intermediates in malleicyprol biosynthesis.
A) Volcano plot analysis comparing pellet extracts of B. thailandensis variants
Pbur and PburΔburA. B) Volcano plot analysis comparing supernatant extracts
of B. thailandensis variants Pbur and PburΔburI (Supplementary Figure 2). C)
Proposed DMSP biosynthesis in B. thailandensis. D) Production of two bis-
malleicyprol (2a, m/z 611.3589) diastereomers in gene inactivation mutants of B.
thailandensis Pbur monitored by UHPLC-MS (EIC in negative ion mode). E)
Genomic alignment of the DMSP assembly line from S. mobaraensis to the bur
biosynthetic gene cluster.
To confirm DMSP as a precursor of malleicyprols, we
performed stable-isotope labeling experiments by chemically
complementing suitable block mutants. Therefore, we required
insight into the molecular basis of DMSP formation in B.
thailandensis. By analogy to one of the established bacterial
DMSP biosynthesis pathways[12] (see ref.[14] for alternative routes
to DMSP) methionine would undergo S-methylation to form S-
methylmethionine (5), decarboxylation, transamination and
oxidation (Figure 2C). In silico analysis of the bur gene locus
revealed candidate genes for a methyltransferase (BurB), a
reference compounds we identified
4 as the zwitterionic
compound dimethylsulfoniopropionate (DMSP; m/z 135.0474;
[M+H]+; Supplementary Figure S1). This finding is intriguing since
DMSP plays a pivotal role in the marine organosulfur cycle,
serving as osmolyte for marine algae and as abundant carbon
and sulfur source for bacteria.[12] It is the precursor of the climate-
relevant gas dimethylsulfide that is emitted at remarkably high
amounts of >107 tons per year into the atmosphere.[13] Despite its
wide distribution, DMSP has thus far not been implicated as a
building block in natural product biosynthesis.
decarboxylase (BurI),
a
transaminase (BurD), and
a
dehydrogenase (BurE) (Figure 2E). Comparison of the deduced
protein sequences to the recently published bacterial DMSP
biosynthetic machinery in Streptomyces mobaraensis[14b] showed
BurD and BurE to be almost identical with their orthologues (96%
and 98%), whereas BurB and BurI are only distantly related to
their S. mobaraensis counterparts (35% and 48%).
To disrupt malleicyprol production, we individually
inactivated each of the four putative DMSP biosynthesis genes in
B. thailandensis Pbur by homologous crossover and replacement
with a resistance cassette. The ΔburD and ΔburE mutants are still
capable of producing the malleicyprol complex, albeit in reduced
amounts (Figure 2D). Possibly, unspecific housekeeping
enzymes take over transamination of sulfonium amine 6 and
oxidation of the instable aldehyde 7.[14b] In contrast, the two main
malleicyprol diastereomers are absent in the ΔburB
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10.1002/anie.202003958
Angewandte Chemie International Edition
Since DMSP accumulates in the ΔburA mutant, we
reasoned that this unusual zwitterionic substrate would be
activated and loaded onto the bur assembly line. In NRPS,
adenylation (A) domains select, activate and load amino acids
onto the assembly line and are thus regarded as gatekeepers.[18]
As bioinformatic substrate predictors[19] failed on BurA-A we
generated a homology model of the A domain. In this way we
noted the absence of the conserved aspartate (D235 in GrsA-A)
present in all α-amino acid activating A domains.[18] Consequently,
BurA-A was expected to select a non-canonical starter unit
lacking the α-amino group usually bound by this aspartate.
According to homology modeling, BurA-A shares important
binding pocket features with the A-domain ATRR-A activating
glycine betaine.[20] In both ATRR-A and BurA-A, the loop carrying
conserved D235 (GrsA-A) has been replaced with a shorter loop
carrying hydrophobic residues (Figure 4A; Supplementary Figure
S5). In another position, both binding pockets have an acidic
residue (D606 in BurA-A) well placed to interact with a positively
charged substrate moiety such as the sulfonium group of DMSP.
To verify whether BurA-A selects and activates DMSP we
cloned and expressed the gene fragment for the A domain (burA-
A) in E. coli BL21(DE3) and purified the His6-tagged protein via
Ni-affinity. We probed the activity of BurA-A with the
MesG/hydroxylamine assay, which monitors pyrophosphate
released during substrate adenylation in a coupled photometric
assay (Figure 4B).[21] From a panel of carboxylic acids, BurA-A
shows highest activity for DMSP. In stark contrast, neither a
(methyltransferase) and ΔburI (decarboxylase) mutants (Figure
2D).
Closer inspection of culture extracts from PburΔburI
revealed enrichment of metabolite 5 (m/z 164.0744 in positive ion
mode) that is identical to the unusual charged amino acid S-
methylmethionine 5 (SMM; Figure 1D and Supplementary Figure
S3). Since 5 cannot be detected in the mutant lacking burB, its
gene product BurB must act in the biosynthetic pathway upstream
of DMSP. BurB’s role in DMSP formation is remarkable as it
belongs to class
containing
V
of the methyltransferase superfamilies
a
SET-domain.[15]
SET-domain-containing
methyltransferases are well studied in eukaryotes and regulate
gene expression through histone lysine methylation.[15] Yet, little
is known about prokaryotic SET-domain methyltransferases, and
natural product modifying methyltransferases have exclusively
been found in class I (Rossmann-like fold) or class III (tetrapyrrole
methylase).[16] To corroborate the function of BurB, we cloned and
overexpressed burB in Escherichia coli and purified the His6-
tagged protein via Ni-affinity. Incubation of purified BurB with L-
methionine (3) and S-adenosylmethionine (SAM) generated SMM
(Figure 3A), which was detected after derivatization with 8 to
compound 9 (Figure 3A). Thus, BurB represents a novel C-S
bond forming enzyme in secondary metabolism.[17]
Having established the key steps to DMSP in B.
thailandensis and with suitable null producers at hand, we
performed chemical complementation. Therefore, we synthesized
13C3-labeled DMSP from 13C3 acrylic acid (10, Figure 3C).
Supplementation of the PburΔburI mutant with 13C3-DMSP not
only restored production of the malleicyprols (see Supplementary
Figure S4) but also enriched 13C in their cyclopropanol residue
(Figure 3B). These results unequivocally confirm DMSP as a key
intermediate in the formation of the malleicyprol warhead.
mixture of the proteinogenic amino acids,
5
nor 3-
(methylthio)propionic acid (11) are activated (Figure 4C).
Apparently, the positively charged sulfonium group enables
substrate binding. Replacing this group with protonated nitrogen
in 12, thus maintaining the positive charge, reduces activity more
than four-fold. In substrate saturation kinetics with DMSP, we
determined
a a KM of 0.15 mM
kcat of 2.4 min-1 and
(Supplementary Figure S7). Altogether, these results indicate that
DMSP is the preferred substrate of BurA-A, making it the first
adenylation domain that incorporates the osmolyte DMSP into a
natural product assembly line.
An in silico analysis of the modular architecture of BurA
suggested that DMSP, once loaded onto the thiolation (T) domain,
would be elongated through Claisen condensation with malonyl-
ACP, and the resulting β-keto intermediate transformed into the
corresponding alcohol by the ketoreductase (KR) domain (Figure
4E). To identify the downstream product of DMSP we
heterologously reconstituted the assembly line. Therefore, we
cloned and expressed burA in E. coli. Only when we employed
IPTG for induction and supplemented DMSP, we detected
production of compound 13 with m/z 179.0742 (positive ion
mode) in XAD16 extracts (Figure 4D). The presence of the same
species in B. thailandensis Pbur (Figure 1B) indicates that
compound 13 is not an artifact generated in E. coli but actually
formed in the intact bur pathway. By HR-LCMS and MS2
comparison with an authentic reference we found that 13 is
identical to the sulfonium acid gonyol.[22] The structure of 13
agrees with our in silico prediction of the biosynthetic steps
mediated by BurA. However, the discovery of this sulfonium
intermediate of the malleicyprol assembly line is surprising, as 13
has been reported as a dominant zwitterion in the marine
dinoflagellate Gonyaulax polyedra.[22] Moreover, 13 is widely
distributed as minor osmolyte in several phytoplankton groups.[23]
Previous studies have identified DMSP and acetate as the
precursors of 13 in G. polyedra,[22, 24] but the enzymes involved in
gonyol biosynthesis have remained unknown. We now report
BurA as the first enzyme involved in a gonyol biosynthetic
Figure 3. DMSP is a precursor of the cyclopropanol warhead of malleicyprol. A)
Transformation of methionine (3) to S-methylmethionine (5; SMM) by BurB and
derivatization with 8; HR-LCMS detection of derivatized SMM; EIC m/z
330.0754 in positive ion mode; top: SMM derivatized with FDNB, middle:
methylation of methionine with BurB, bottom: heat-inactivated BurB. B) Mass
spectra of native bis-malleicyprol (top) and of 13C-enriched bis-malleicyprol
(bottom) C) Synthesis of 13C-labeled DMSP and subsequent complementation
of B. thailandensis PburΔburI leads to incorporation of the C3 unit into 2.
This article is protected by copyright. All rights reserved.

10.1002/anie.202003958
Angewandte Chemie International Edition
PKS-NRPS hybrid as a gonyol synthetase allows for genomics-
based identification of ecologically relevant producer strains.
From a biosynthetic perspective DMSP is noteworthy as a novel
PKS primer unit,[26] and the sulfonium-accepting adenylation
domain BurA-A is an important addition to the synthetic biology
toolbox that opens up new possibilities for engineering
polyketides and nonribosomal peptides.
pathway, and unexpectedly, it is a modular NRPS-PKS hybrid.
We reason that the sulfonium group represents a leaving group,
likely as dimethylsulfide that enables cyclopropanol formation
downstream of BurA. The enzymes and mechanisms involved in
the cyclization are the subject of ongoing studies.
Acknowledgements
We thank A. Perner for LC-MS measurements. Financial support
by the DFG (SFB 1127 ChemBioSys, and Leibniz Award to C. H.)
and the European Regional Development Fund (ERDF)
(MassNat) is gratefully acknowledged. J. F. acknowledges
financial support by the SMART BIOTECS alliance between the
Technische Universität Braunschweig and the Leibniz Universität
Hannover, supported by the Ministry of and Culture (MWK) of
Lower Saxony, Germany. H. K. received a fellowship from the
Daimler and Benz foundation.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Biosynthesis · DMSP · Mass spectrometry · NRPS ·
Virulence factors
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In summary, we have uncovered crucial steps in the
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Angewandte Chemie International Edition
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10.1002/anie.202003958
Angewandte Chemie International Edition
COMMUNICATION
Sulfur cycle meets pathogen.
Sulfonium acids known from global
sulfur cycles were elucidated as
the biosynthetic origin of the
cyclopropanol warhead of
F. Trottmann, K. Ishida, J. Franke, A.
Stanišić, M. Ishida-Ito, H. Kries, G.
Pohnert, C. Hertweck*
Page No. – Page No.
malleicyprols, virulence factors of
human-pathogenic bacteria. The
pathway involves a specialized S-
methyl transferase to form DMSP,
which is activated by a zwitterion-
specific adenylation domain and
transformed into the key
Sulfonium Acids Loaded onto
Unusual Thiotemplate Assembly
Line Constitute Cyclopropanol
Warhead of a Burkholderia Virulence
Factor
intermediate gonyol.
Institute and/or researcher Twitter usernames: @c_hertweck; @Hertweck_Lab; @KriesLab; @LeibnizHKI
This article is protected by copyright. All rights reserved.