Biosynthesis of the Enterotoxic Pyrrolobenzodiazepine Natural Product Tilivalline

Article abstract of DOI:10.1002/anie.201707737

The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is part of the intestinal microbiota and the pyrrolobenzodiaze

Full text of DOI:10.1002/anie.201707737

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Accepted Article
Title: Biosynthesis of the enterotoxic pyrrolobenzodiazepine natural
product tilivalline
Authors: Elisabeth Dornisch, Jakob Pletz, Ronald A. Glabonjat, Florian
Martin, Christian Lembacher-Fadum, Margit Neger, Christoph
Hoegenauer, Kevin Francesconi, Wolfgang Kroutil, Klaus
Zangger, Rolf Breinbauer, and Ellen L. Zechner
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201707737
Angew. Chem. 10.1002/ange.201707737
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10.1002/anie.201707737
Angewandte Chemie International Edition
COMMUNICATION
relevant phenotypes.[2-6] The enteric bacterium K. oxytoca
presents a striking example. In some patients taking penicillin
antibiotics, rapid overgrowth of this organism results in antibiotic-
associated hemorrhagic colitis (AAHC).[7] We showed in
Biosynthesis of the enterotoxic
pyrrolobenzodiazepine natural
product tilivalline
Elisabeth Dornisch1, Jakob Pletz2,3, Ronald A.
Glabonjat3, Florian Martin1, Christian Lembacher-
Fadum2, Margit Neger1, Christoph Högenauer4,
Kevin Francesconi3, Wolfgang Kroutil3, Klaus
Zangger3, Rolf Breinbauer2,5* and Ellen L.
Zechner1,5*
previous
work
that
the
indol-3-yl
substituted
pyrrolobenzodiazepine (PBD) tilivalline (1) produced by K.
oxytoca was required for the pathogenesis of colitis in an animal
model of AAHC.[8] Natural product PBDs form a family of
antitumor antibiotics produced by Gram-positive soil bacteria.[9]
Synthesis of a natural PBD by a Gram-negative resident of the
gut is thus surprising. We identified the toxin biosynthetic gene
cluster by genetic mutation and localized the region to a unique
pathogenicity island on the genome of cytotoxic K. oxytoca
strains.[8]
Here we report the biosynthesis of tilivalline and characterize
a second PBD monomer generated by this pathway, tilimycin,
with stronger cytotoxic properties. The toxin biosynthetic genes
carried by the pathogenicity island are organized in two operons
(Figure S1). BLAST analysis identified genes involved in
synthesis of aromatic amino acids and related aromatic
Abstract: Nonribosomal enterotoxin tilivalline was the first natural
pyrrolobenzodiazepine linked to disease in the human intestine.
Since the producing organism Klebsiella oxytoca is part of the
intestinal microbiota and the pyrrolobenzodiazepine causes the
pathogenesis of colitis it is important to understand the biosynthesis
and regulation of tilivalline activity. Here we report the biosynthesis
of tilivalline and show that this non-ribosomal peptide assembly
pathway initially generates a simple pyrrolobenzodiazepine with
cytotoxic properties named tilimycin. Tilivalline results from a non-
enzymatic spontaneous reaction of tilimycin with biogenetically
generated indole. Through a chemical total synthesis of tilimycin we
could corroborate the predictions made about the biosynthesis.
Production of two cytotoxic pyrrolobenzodiazepines with distinct
functionalities by human gut resident Klebsiella oxytoca has
important implications for intestinal disease.
compounds:
a
4-hydroxyphenyl acetate-3-monoxygenase
(hmoX), a 2-amino-2-deoxy-isochorismate synthase (adsX), an
isochorismatase (icmX), a 2,3-dihydro-dehydrogenase (dhbX)
and
a
2-keto-3-deoxy-D-arabino-heptolosonate phosphate
synthase (aroX). The NRPS-operon contains tilivalline specific
non-ribosomal peptide synthases npsA, thdA and npsB. Our
earlier mutagenesis studies showed that cluster genes aroX,
npsA+thdA and npsB, and a 3-dehydroquinate synthase (aroB)
at
a distant genomic locus are essential for tilivalline
biosynthesis.[8] However, no genes involved in forming the indole
ring at the C11-position of the tilivalline structure were apparent.
K. oxytoca are indole producers predicted to use
a
tryptophanase to catalyze cleavage of L-tryptophan to indole,
pyruvate, and ammonium.[10] We inactivated the putative
tryptophanase gene (tnaA) of K. oxytoca AHC-6. Wild type (WT)
AHC-6 and the ∆tnaA mutant were cultivated in vitro and the
presence of tilivalline in culture broth sampled during 48 h of
growth was followed by HPLC-MS. In contrast to the K. oxytoca
WT culture no tilivalline was detected for the ∆tnaA mutant (Fig.
1A). Unexpectedly, however, spent medium of the mutant ∆tnaA
showed cytotoxic activity on HeLa cells comparable to the
tilivalline-producing WT strain (Fig. 1B), which suggested that
the ∆tnaA mutant might produce a different cytotoxic secondary
metabolite. By extraction of the culture broth of the ∆tnaA mutant
with butanol and subsequent purification via preparative HPLC,
two so far undescribed small molecule metabolites were isolated
and spectroscopically characterized. Both metabolites have
identical molecular weight (234.3 g/mol; C12H14N2O3). The first
metabolite, which we named tilimycin (2), shares with tilivalline
the same pyrrolo[2,1-c][1,4]benzodiazepine motif but has an
hydroxyl group instead of the indole ring at the C11 position
resulting in a hemiaminal moiety, which is also responsible for
the low stability of 2 (Fig. 1C). Tilimycin (2)[11] is more cytotoxic
to HeLa cells (IC50=2.6 µM) than tilivalline (1) (IC50=14.5 µM; Fig.
S6). The second new metabolite was isolated from culture broth
of both WT and ∆tnaA mutant strains after incubation for >24 h.
This product, which we named culdesacin (3), belongs to the
class of pyrrolo[1,2-b]isoquinolin-5(1H)-ones. Unlike 1 and 2, 3
shows no cytotoxic activity (Fig. S6).
Antibiotic therapy disrupts the human intestinal microbiota.
Shifts in microbial communities have been correlated to the
pathogenesis of many disorders including metabolic diseases,
cancer, and disorders of the liver, bowel and lung.[1],[2] Given the
vast number of potentially bioactive substances produced by the
gut microbiota one of the greatest challenges facing researchers
is to link a specific organism and its metabolites to a particular
pathological outcome for the host.[3],[4] However, microbial small
molecules have been shown to cause some of these clinically
1 Elisabeth Dornisch, Margit Neger, Florian Martin, Prof. Dr. Ellen L. Zechner
Institute of Molecular Biosciences
University of Graz
Humboldtstrasse 50/I, 8010 Graz, Austria
E-mail: ellen.zechner@uni-graz.at
2 Dr. Jakob Pletz, Christian Lembacher-Fadum, Prof. Dr. Rolf Breinbauer
Institute of Organic Chemistry
Graz University of Technology
Stremayrgasse 9, 8010 Graz, Austria
E-mail: breinbauer@tugraz.at
3 Dr. Jakob Pletz, Ronald A. Glabonjat, Prof. Dr. Kevin Francesconi, Prof. Dr.
Wolfgang Kroutil, Prof. Dr. Klaus Zangger
Institute of Chemistry
University of Graz
Heinrichstrasse 28 & Universitätsplatz 1, 8010 Graz, Austria
4 Dr. Christoph Högenauer
Division of Gastroenterology and Hepatology, Department of Internal Medicine
Medical University of Graz
Auenbruggerplatz 15, 8036 Graz, Austria
5 BioTechMed-Graz
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10.1002/anie.201707737
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COMMUNICATION
This could be verified by HPLC detection of tilivalline after
synthetic tilimycin and indole were added to medium lacking
bacteria or enzyme activity (Fig. S4). These experiments
[12]
showed that spontaneous conversion of tilimycin (2)
to
culdesacin (3) occurs and that 2 reacts with indole to form
tilivalline (1) as the natural trans-isomer in the absence of
enzyme activities (see Figure S8).
Having identified tilimycin (2) as the precursor for tilivalline
(1) we next addressed the biosynthesis of 2. The adenylation (A)
domains of NpsA and NpsB were analyzed for substrate-
activating specificity. The NpsA/ThdA module is predicted to
accept anthranilate-substrates and NpsB L-proline.[13] The aroX-
dhbX-icmX-adsX-hmoX operon may provide the anthranilic
substrate via enzymes related to the shikimate and chorismate
pathways (Scheme 2) similar to biosynthesis of the analogous
PBDs anthramycin or tomaymycin.[14] The 2-keto-3-deoxy-D-
arabino-heptolosonate phosphate synthase AroX is independent
of amino acid feed-back regulation like its counterpart TomC in
tomaymycin synthesis.[14-15] AroX and the 3-dehydroquinate
synthase AroB are involved in chorismate synthesis and are
essential for tilivalline biosynthesis.[8] Chorismate can be
converted by AdsX to 2-amino-2-deoxy-isochorismate, as was
shown for analogues TomD in tomaymycin biosynthesis and
PhzE in phenazine biosynthesis.[15],[16] We asked whether 3-
hydroxyanthranilic acid (3HAA) is the substrate of NpsA/ThdA.
Two pathways for production of this precursor from 2-amino-2-
deoxy-isochorismate (ADIC) are conceivable (Scheme 2). In the
first pathway, drawn on phenazine biosynthesis, ADIC is
converted to trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA)
Figure 1: A) Time-dependent formation of 1 in conditioned medium of K.
oxytoca WT and K. oxytoca ∆tnaA, as measured by HPLC-ESI-MS (signal
counts at m/z 334.2); B) Cytotoxicity to HeLa cells of medium conditioned by K.
oxytoca AHC-6 WT and K. oxytoca AHC-6 ∆tnaA over time as indicated; C)
Chemical structures of tilivalline (1), tilimycin (2), and culdesacin (3); D) HPLC-
ESI-MS chromatograms of synthetic standards of tilivalline (1) – m/z 334.2,
tilimycin (2) and culdesacin (3) – m/z 235.1; E) HPLC-ESI-MS chromatograms
(red – m/z 235.1 and blue – m/z 334.2) of butanol extracts of 24 h cultures as
indicated.
The three metabolites 1-3 were detected by HPLC-ESI-MS in
butanol extracts of conditioned medium from K. oxytoca WT but
not from the toxin-negative K. oxytoca ∆npsA, where the specific
non-ribosomal peptide synthase NpsA is absent. The ∆tnaA
mutant produced only
2
and 3. Importantly, tilivalline
biosynthesis was restored in the mutant by genetic
complementation (K. oxytoca AHC-6 ∆tnaA [pTnaA]) or addition
of 500 µM indole (Fig. 1E).
These findings led us to propose that tilivalline (1) may form
spontaneously by nucleophilic addition of indole made available
by tryptophanase at an imine intermediate in situ generated from
2 (Scheme 1).
Scheme 2. Proposed biosynthetic pathways of tilimycin (2) via 3-
hydroxyanthranilic acid (substrate of NpsA/ThdA) and L-proline (substrate of
NpsB). AroX: 2-keto-3-deoxy-D-arabino-heptolosonate phosphate (DHAP)
synthase; AroB: 3-dehydroquinate synthase; AdsX: 2-amino-2-deoxy-
Scheme 1. Proposed chemical reactions of tilimycin (2) to culdesacin (3) via
spontaneous ring opening and to tilivalline (1) via a nucleophilic attack of free
indole, released by the tryptophanase (TnaA)-catalyzed cleavage of L-
tryptophan.
isochorismatase;
IcmX:
isochorismatase;
DhbX:
2,3-dihydro-2,3-
dihydroxybenzoate dehydrogenase; HmoX: 4-hydroxyphenyl acetate-3-
monoxygenase; NpsA/ThdA: non-ribosomal peptide synthase; NpsB: non-
ribosomal peptide synthase.
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10.1002/anie.201707737
Angewandte Chemie International Edition
COMMUNICATION
by the isochorismatase IcmX, which is 40% identical to
isochorismatase PhzD of Pseudomonas aeruginosa PAO1
(NP_252903.1).[17] The oxidation of DHHA to 3HAA could be
catalyzed by 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase
DhbX (Scheme 2, route a). The second pathway draws on
tomaymycin biosynthesis where chorismate conversion to
AA. K. oxytoca WT and K. oxytoca ∆npsA were included as
controls. The conditioned medium of these experiments was
then extracted and analyzed by HPLC for the amounts of 1-3.
Strains lacking NpsA, AroB, AroX, AdsX, IcmX or DhbX were
functionally deficient for the biosynthesis of all three metabolites.
By contrast deletion of hmoX and the genomic homologue
(∆∆hmoX) had no effect. The observed deficiencies were
complemented chemically by addition of 3HAA to culture
medium in every case, except for the ∆npsA control strain. The
addition of AA to mutant cultures did not restore production of 2
or 1 but instead their respective deoxy-derivatives 9-deoxy-
tilimycin (4) and 9-deoxy-tilivalline (5) (Fig. 2A-B). All five
metabolites could be detected after addition of AA to the WT
strain, whereas none were observed for control strain K. oxytoca
∆npsA in all cases. Deoxy-culdesacin was not detected,
presumably due to the lack of the aromatic hydroxyl group,
which can be considered essential for nucleophilic attack of the
arene at the aldehyde electrophile.
anthranilic acid (AA)
requires
the 2-amino-2-deoxy-
isochorismate synthase (TomD) and the anthranilate synthase
(TomP).[15] Hydroxylation of AA to 3HAA could be catalyzed by a
4-hydroxyphenylacetate-3-monoxygenase as it was shown for
GTNG_3160 in Geobacillus thermodenitrificans NG80-2
(Scheme 2, route b).[18] hmoX and a second homologue on the
AHC-6
genome
encode
4-hydroxyphenylacetate-3-
monoxygenases 32% identical to GTNG_3160. To date the K.
oxytoca genome lacks an annotated anthranilate synthase.
To test these pathways, K. oxytoca AHC-6 single gene
mutants ∆aroB, ∆aroX, ∆adsX, ∆icmX, ∆dhbX were cultured in
CASO medium or medium supplemented with either 3HAA or
Figure 2. Feeding experiments with K. oxytoca mutant strains confirm tilimycin (2) synthesis via 3-hydroxyanthranilic acid (3HAA). The addition of synthetic
anthranilic acid (AA) led to the mutasynthesis of 9-deoxy-tilimycin (4) and 9-deoxy-tilivalline (5). A) HPLC-ESI-MS-chromatograms (red – m/z 235.1, blue – m/z
334.2, green – m/z 219.1, violet – m/z 318.2) of n-butanol extracts of conditioned medium (24h culture) from K. oxytoca AHC-6 WT, ∆npsA, ∆aroB, ∆adsX, ∆icmX,
∆dhbX, ∆∆hmoX grown in medium (left column), or medium supplemented with 3HAA (middle column) or with AA (right column); B) Chemical structures of 4 and
5; C) HPLC-ESI-MS-chromatograms of synthetic standards of (1) – m/ 334.2 (blue), (2) and (3) – m/235.1 (red), (4) – m/z 219.1 (green), (5) – m/z 318.2 (violet);
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10.1002/anie.201707737
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COMMUNICATION
In conclusion, by a combination of metabolite profiling,
genetic deletion experiments, and complementation experiments
with synthetically prepared intermediates we established the
biosynthetic pathway of tilimycin (2) and tilivalline (1) (Scheme
3). 3HAA, synthesized from chorismate by enzymes from the
pathogenicity island, is processed by a non-ribosomal peptide
synthase to N-acylprolinal 6. The subsequent ring closure to
tilimycin (1) as well as the introduction of the indole moiety does
not require a specific enzyme but can occur spontaneously
through the intrinsic reactivities of the pertinent reaction
intermediates. We could show that the biosynthetic pathway can
Finally, the results of this study are particularly significant in
the physiological context. Since feces of healthy humans usually
contain 1.0 mM to 4.0 mM indole,[20] we anticipate that both 1
and 2 are present in the intestine and exert distinct cytotoxic
functionalities that contribute specifically to AAHC and possibly
other disorders.
Acknowledgements
We thank Hansjörg Weber for recording NMR spectra and Pål W.
Wallace for high resolution mass measurements. Research was
funded by the Austrian Science Fund (FWF) W901DK Molecular
Enzymology (to ELZ, RB, WK and KZ), BioTechMed-Graz
Secretome Flagship (to ELZ and RB), and NAWI Graz.
be
exploited
for
mutasynthesis
of
unnatural
pyrrolobenzodiazepine through the addition of anthranilic acid
and indole derivatives.
Keywords: biosynthesis, natural products,
pyrrolobenzodiazepines, nonribosomal peptide, gut microbiota
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Scheme 3. Complete biosynthesis of tilivalline (1) via tilimycin (2). After
binding and activation of 3-hydroxanthranilic acid and L-proline to the non-
ribosomal peptide synthases NpsA/ThdA and NpsB the reductive release to an
open N-acylprolinal (6) occurs. From the final product of enzymatic synthesis
either tilimycin (2) or culdesacin (3) is formed. Tilimycin (2) can be further
converted to tilivalline (1) after the nucleophilic attack of free indole, released
by the bacterial tryptophanase (TnaA) after enzymatic cleavage of L-
tryptophan.
[10] J. N. Maslow, S. M. Brecher, K. S. Adams, A. Durbin, S. Loring, R. D.
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The PBD family of potent cytotoxic agents has been
extensively investigated for use in systemic chemotherapy.[5]
PBD monomers target purine-guanine-purine motifs in the minor
groove of DNA. Once situated in the minor groove, an aminal
bond is formed between the C11 position of the PBD and the N2
of guanine. Presence of the indole ring at the C11 position of
tilivalline (1) blocks this activity, but tilimycin (2) is expected to
belong to the DNA-interacting antitumor agents. Thus K. oxytoca
is able to produce two PBD cytotoxins with distinct functionalities
depending on the availability of indole.
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approach to introduce structural modifications that enhance
biological activity and potency of anticancer analogues.
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COMMUNICATION
COMMUNICATION
Klebsiella
oxytoca
enterotoxin
Elisabeth Dornisch 1, Jakob Pletz 2,3
,
Ronald A. Glabonjat3, Florian Martin1,
Christian Lembacher-Fadum2, Margit
Neger1, Christoph Högenauer , Kevin
Francesconi3, Wolfgang Kroutil3, Klaus
Zangger3, Rolf Breinbauer2,5, * and
tilivalline was the first natural
pyrrolobenzodiazepine linked to
disease in the human intestine. We
report the biosynthesis of tilivalline
and show that this non-ribosomal
peptide assembly pathway initially
Ellen L. Zechner1,5
*
generates
pyrrolobenzodiazepine
a
cytotoxic
named
Page No. – Page No.
tilimycin. Tilivalline results from a
non-enzymatic spontaneous reaction
of tilimycin with biogenetically
generated indole.
Biosynthesis of the enterotoxic
pyrrolobenzodiazepine natural
product tilivalline
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