A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation

Article abstract of DOI:10.1021/jacs.1c04996

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O₂into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphicBurkholderiaplantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marineRhodobacteraceae(e.g.,Phaeobacter inhibens).

Full text of DOI:10.1021/jacs.1c04996

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Article
A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis
via Coenzyme A‑Ester Oxygenolysis and Ring Epoxidation
Ying Duan,⊥ Marina Toplak,⊥ Anwei Hou,⊥ Nelson L. Brock, Jeroen S. Dickschat,* and Robin Teufel*
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ABSTRACT: Bacterial tropone natural products such as
tropolone, tropodithietic acid, or the roseobacticides play crucial
roles in various terrestrial and marine symbiotic interactions as
virulence factors, antibiotics, algaecides, or quorum sensing signals.
We now show that their poorly understood biosynthesis depends
on a shunt product from aerobic CoA-dependent phenylacetic acid
catabolism that is salvaged by the dedicated acyl-CoA dehydrogen-
ase-like flavoenzyme TdaE. Further characterization of TdaE
revealed an unanticipated complex catalysis, comprising substrate
dehydrogenation, noncanonical CoA-ester oxygenolysis, and final
ring epoxidation. The enzyme thereby functions as an archetypal
flavoprotein dioxygenase that incorporates both oxygen atoms
from O2 into the substrate, most likely involving flavin-N5-
peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous
decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused
by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-
containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).
ring-cleaving enoyl-CoA hydratase (ECH), either as a
standalone enzyme or as part of the bifunctional fusion
protein PaaZ, which typically comprises a C-terminal ECH and
an N-terminal aldehyde dehydrogenase (ALDH) domain.5
The formed semialdehyde intermediate 3 is highly reactive and
could not be observed or captured by derivatization so far.5
Either this aldehyde group is immediately oxidized to the more
stable carboxylate (8) by the PaaZ-ALDH domain or a
separate ALDH along the downstream steps of the paa
catabolon that is followed by β-oxidation-like steps,3 or a rapid
spontaneous intramolecular Knoevenagel condensation to
shunt product 4 occurs (Figure 1).
Some bacteria appear to have developed mechanisms to
boost 4 formation, as exemplified by P. inhibens, which encodes
a PaaZ homologue with an ALDH domain that lacks the
catalytic residues for aldehyde oxidation and thereby likely
drives its accumulation.15 Compound 4 may then be
converted, for example, into tropolone (9) and hydroxytropo-
lones (e.g., 10) by Burkholderia spp. (including plant
INTRODUCTION
■
Bacterial natural products that feature a non-benzenoid
aromatic tropone core (1, Figure 1) are of environmental
and pharmaceutical importance and are produced by
numerous marine and terrestrial bacteria.1,2 Their biosynthesis
was previously linked to phenylacetic acid (paa) (2)
degradation,3,4 in which a reactive semialdehyde intermediate
(3) undergoes an intramolecular condensation reaction to
yield the shunt product 2-hydroxycyclohepta-1,4,6-triene-1-
formyl-CoA (4), which was hypothesized to be the universal
tropone precursor based on its structural features (Figure 1).5
Compound 2 is typically obtained from the environment and
may also arise from the catabolism of other aromatic
compounds such as styrene, ethylbenzene, or phenylalanine.3,6
In addition, 2 can be generated from the anabolic shikimate
pathway product phenylpyruvic acid, which is likely a common
strategy for tropone natural product forming bacteria.5,7 The
formation of 4 from 2 typically requires four enzymes. First, 2
is activated by the phenylacetate:CoA ligase PaaK, which
generates phenylacetyl-CoA (5).8−10 Alternatively, 5 is directly
produced from phenylpyruvic acid, as previously shown for
Phaeobacter inhibens.6 Compound 5 is then epoxidized and
dearomatized to 1,2-epoxyphenylacetyl-CoA (6) by the di-
iron-dependent multicomponent monooxygenase
PaaABCE,3,11,12 before the isomerase PaaG converts 6 into
(Z)-2-(oxepin-2(3H)-ylidene)-acetyl-CoA (“oxepin-CoA”,
7).3,13,14 The α,β-unsaturated 7 is further processed by a
Received: May 17, 2021
Published: July 1, 2021
© 2021 The Authors. Published by
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Figure 1. Bacterial 2 catabolic pathway and generation of the proposed universal tropone precursor 4. Catabolic steps generate reactive 3, which is
oxidized at C8 to the stable carboxylic acid 8, before final β-oxidation-like steps generate central metabolites (pathway A, gray arrows).
Alternatively, 3 spontaneously cyclizes to 4 via an intramolecular Knoevenagel condensation (pathway B, black arrows). TdaE then converts 4 into
18 (red dashed box), which is prone to undergo decarboxylation to natural product 9. In addition, 18 is likely the precursor for sulfur-containing
tropone natural products such as 11, 14, and 15. Oxygen atoms shown in red and blue indicate incorporation from O2 and H2O, respectively
(based on 18O-isotope labeling experiments). For details on the flavin-dependent TdaE catalysis, see text and Figure 5. Examples of mature tropone
natural products and selected bioactivities are shown in the black box. The carbon numbering for all compounds is according to 3.
pathogens such as B. plantarii),16−18 Pseudomonas donghuen-
sis,19,20 and Streptomyces spp.21 In addition, 4 is most likely the
precursor for more complex sulfur-containing derivatives, i.e.,
tropodithietic acid (11) (and its tautomers troposulfenin (12)
and thiotropocine (13))4,15,22 and the roseobacticides A−G23
(e.g., roseobacticide A; 14) from predominant marine
Rhodobacteraceae (Roseobacter spp., Phaeobacter spp., or
Pseudovibrio spp. among others), as well as a sulfur-bridged
tropolone dimer (ditropolonyl sulfide) (15) from the human
pathogen Burkholderia cenocepacia,24 which can infect cystic
fibrosis patients. Many of these compounds are critical for
symbiotic interactions; for example, 1125,26 is produced by
bacteria that often live associated with marine invertebrates
(sponges, tunicates, soft and stony corals, tube worms,
shellfish, among others) and algae1,26 and likely serves as an
antibiotic that protects the host organisms against pathogens
such as Vibrio spp.. Interestingly, 11 was also shown to act as a
quorum sensing signal that triggers major changes in bacterial
gene expression.22 Similarly, tropolones play important roles in
symbiotic interactions, most notably the antagonism of
Burkholderia spp. such as B. plantarii that cause bacterial
panicle blight in rice plant seedlings and thus pose a threat to
global rice production.16,27 It was shown that 9 is the key
virulence factor of B. plantarii and likely deprives the plants of
essential iron via chelation.28,29
antibiotic 15 from B. plantarii and B. cenocepacia have not been
reported to date.
We now show that 4 indeed serves as a central precursor for
structurally distinct bacterial tropone natural products and as a
substrate for the key flavoenzyme TdaE, which is encoded by
the previously reported 11-BGCs of marine Rhodobacteraceae
and by the newly identified putative BGCs for the generation
of 9 and 15 in Burkholderia spp. Our studies include the
detailed analysis of the reactions catalyzed by heterologously
produced TdaE homologues and the probing of the enzyme
mechanism using LC-HRMS and 18O isotope labeling
experiments among other techniques, which reveal a surprising
prototypal dioxygenase functionality. The rapid spontaneous
decomposition of the unstable TdaE product strongly
hampered its structure elucidation, which could only be
achieved through the use of 13C-labeled precursors and by a
combination of chemical derivatization and comparison to an
enantioselectively synthesized reference compound. Ulti-
mately, the reactive TdaE product either spontaneously
forms 9 or is likely further transformed into 11 and other
sulfur-containing tropone natural products.
RESULTS
■
Burkholderia spp. Harbor tdaE Homologues for the
Production of Tropolone and Ditropolonyl Sulfide. To
identify putative BGCs of tropone natural products in
pathogenic Burkholderia species, protein BLAST searches
were conducted using proteins as queries that were previously
associated with tropone biosynthesis in addition to enzymes
involved in producing aromatic precursors de novo via the
shikimate pathway.1 The search was focused on B. plantarii and
B. cenocepacia strains, reported to produce 9 and 15,
respectively. Initially, a predicted thioesterase and two
flavoprotein monooxygenases (FPMOs) from the recently
As of yet, the enzymatic step that links bacterial tropone
biosynthesis with 2 catabolism has not been verified, and
downstream biosynthesis consequently remains poorly under-
stood.1,30 Biosynthetic gene clusters (BGCs) were previously
reported for 11 (also required for the roseobacticides)4,31,32
from Phaeobacter spp. and for 10 from Streptomyces spp.,21 but
direct evidence for the roles of the encoded enzymes is
lacking.1 In contrast, BGCs for the formation of toxic 9 and the
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Figure 2. Proposed biosynthetic gene clusters for bacterial tropone natural products with the encoded proteins shown on top (paa catabolic genes
are located elsewhere in the genome, cf. Figure S1). Top: Verified tda gene cluster from P. inhibens for production of 11 that contains a second copy
of paaZ encoding an enzyme with a dysfunctional ALDH domain. The newly identified putative gene clusters for 9 and 15 formation from B.
plantarii and B. cenocepacia are shown below. Genes predicted to encode enzymes involved in supplying pathway precursors are shown in gray with
the exception of patB (orange) and tdaB (yellow), which presumably encode enzymes for formation and incorporation of the sulfur precursor,
respectively. Other genes encoding proposed enzymes involved in downstream processing of 4 toward mature tropone natural products are also
color coded individually (tdaE is shown in red). Genes encoding transcriptional regulators, putative transporters, and proteins of unknown function
are shown in white. The gene encoding a putative decarboxylase of B. plantarii that was investigated in this work is marked with an asterisk. See
Tables S1−S3 for details and accession numbers of the predicted proteins.
Figure 3. Time course of 4 conversion into 18 by TdaEPi and analysis of 18O incorporation into compound 18 by LC-HRMS. (A) Time course for
the conversion of 4 via 16 into 18, as determined by RP-HPLC analysis. The pink curves correspond to the assays containing TdaEPi,
demonstrating that after a rapid conversion of 4 into 16, compound 18 is produced. The control reaction (black curves) only shows the
spontaneous oxidation of 4 to 16 without formation of 18. (B) HPLC chromatograms (at 300 nm) corresponding to the time course graphs shown
in A, with the substrate 4, the reaction intermediate 16, and the final product 18 indicated by gray, blue, and purple lines, respectively. The peak
with a retention time of 9.5 min (at t(0 min), prior to the addition of TdaEPi) forms spontaneously from 4 and likely represents a tautomer that is
converted into 16 by TdaE. (C) MS fragmentation pattern of compound 18 (negative ion mode) generated in TdaE assays in the presence of
either 16O2 (left) or 18O2 (right), in which the in-source fragmentation of 18 to 9 by decarboxylation can be observed.
reported Streptomyces spp. were used as queries. Both these
enzymes are essential for the production of hydroxytropolones
such as 10,21 presumably by mediating CoA-thioester cleavage
as well as ring oxidation and hydroxylation.1,21 However, no
genomic regions encoding such enzymes were found; instead,
homologues of genes from 11 biosynthesis of P. inhibens were
identified in both B. plantarii and B. cenocepacia Bp8974 as part
of putative BGCs. These genes encoded enzymes of the
shikimate pathway as well as homologues of the predicted
flavoenzyme TdaE from 11 biosynthesis that was suggested to
be involved in the downstream processing of 4 (Figure 2).1,15
TdaE has low similarity to flavin-dependent acyl-CoA
dehydrogenases (ACADs) and was previously proposed to
catalyze the two-electron oxidation of the dihydrotropone
moiety of 4.15 In addition, consistent with the structure of 15,
the BGC of B. cenocepacia encoded homologues of putative
sulfur precursor-synthesizing (PatB) and -incorporating
(TdaB) enzymes that were previously found in the BGCs of
11 producers (Figure 2).33
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TdaE Catalysis Involves Initial Substrate Dehydro-
genation. To investigate the role of TdaE in the biosynthesis
of sulfur-containing tropone derivatives and of tropolone, the
TdaE homologues encoded by the BGCs of P. inhibens
(TdaEPi; NCBI accession ID: WP_014881725.1) and B.
plantarii (TdaEBp; NCBI accession ID: WP_042624079.1)
were heterologously produced and purified. Both enzymes
could eventually be obtained in soluble form (Figures S2−S4)
with an N-terminal maltose-binding protein (MBP) for TdaEPi
and an N-terminal GB1 (subunit B of protein G) as well as a
C-terminal polyhistidine tag for TdaEBp (both tags were
required to obtain soluble and stable enzyme). After protein
purification, TdaEPi was obtained almost colorless, whereas
TdaEBp was weakly yellow due to a loosely bound FAD
cofactor (Figures S5, S6) (based on c280/c450 stoichiometry
protein:FAD ca. 5:1). To investigate a possible biosynthetic
role of TdaE, in vitro enzyme assays were established in which
chemically synthesized 5 was used as a substrate for
heterologously produced PaaABCE, PaaG, and PaaZ-E256Q
(a PaaZ variant with inactive ALDH domain that reroutes the
paa catabolic pathway to the formation of 4).5 Addition of
either TdaEPi or TdaEBp further converted 4 into a new
compound that also formed spontaneously at much lower rates
and was retained in the aqueous phase after organic extraction
with ethyl acetate (EtOAc). LC-HRMS analysis supported the
envisaged two-electron oxidation reaction of 4 to 16 (MH+
m/z 900.145) (Figure S7). To verify the proposed structure of
16, the CoA ester was hydrolyzed with heterologously
produced thioesterase PaaY, which normally salvages trapped
CoA from the inhibitory shunt product 4 in 2-degrading
bacteria.11 Following the extraction with EtOAc, the hydrolysis
product was identified as tropone-2-carboxylate (17) (MH−
m/z 149.024) as it exhibited the same retention time as well as
identical UV−vis and HRMS spectra compared to a chemically
synthesized standard25 (Figure S8). Notably, 16 also formed
spontaneously from 4 in TdaE-free control reactions, albeit
significantly slower (Figure 3A,B). These findings confirmed
the TdaE dehydrogenase functionality, consistent with the
homology to ACADs. However, following the initial
accumulation of 16 in the TdaE assays, a rapid decrease of
this compound was observed, suggesting that 16 may only
represent an intermediate in the TdaE-catalyzed reaction
(Figure 3A,B). To investigate this, TdaE assays were
scrutinized over time, revealing the generation of a distinct
final product that could not be observed in control assays
(Figure 3A,B; Figures S9, S10).
infra for a gene deletion experiment). To elucidate the
structure of the final TdaE product 18, large-scale enzymatic
assays were conducted; however, the compound proved
unstable and slowly decomposed in the enzyme assays (Figure
S14) and more rapidly during NMR sample preparation into a
volatile compound that was easily lost in concentration steps.
Several trials to isolate the TdaE product in sufficient amounts
failed, precluding its structure elucidation by standard NMR-
based methods (only partial 1D and 2D NMR spectra showing
signals for five hydrogens, but only two signals for olefinic/
aromatic CH groups could be obtained; Figures S15 and S16).
Therefore, an isotopic labeling strategy was employed to
enhance the missing 13C NMR signals for elucidation of the
structure of the TdaE product. For this purpose, (13C8)-2 was
chemically synthesized according to Scheme S1 in the
Materials and Methods (for NMR spectra of intermediates
and (13C8)-2 cf. Figures S17−S25) and enzymatically
converted into (13C8)-5 by PaaK to serve as a substrate for
the enzyme assay with PaaABCE, PaaG, PaaZ-E256Q, and
TdaE. The product was enriched by RP-HPLC and analyzed
by 1D and 2D 13C NMR spectroscopic methods. During
measurement in CD3CN, however, the labeled compound
once more gradually degraded under accumulation of a
breakdown product, showing cross-peaks in the 13C,13C-
COSY NMR (Figure S26) only between four sp2 carbons
(one quaternary and three CH groups). This spin system was
reflected by 13C−13C couplings in the 13C NMR spectrum
(Figure S15) in line with the pseudosymmetrical (C2v)
structure of (13C7)-9. In this compound the two halves can
interconvert by fast keto−enol tautomerism, making them
identical on the NMR time scale. The identity of (13C7)-9 was
confirmed by spiking the NMR sample with commercially
available unlabeled 9 (Figure S27).
For a full understanding of the formation of 9, the
identification of its unstable precursor 18 was required.
Enzymatic conversion of (13C8)-2 and commercially available
(1,2-13C2)-2 with optimization of the workup procedure and
immediate NMR measurements of the freshly obtained
samples allowed the identification of the final TdaE products
by 13C NMR, 13C,13C-COSY NMR, and HSQC (Figures S28−
S31 and Table S4) through the strong enhancement of all 13C-
based NMR signals as (13C8)- and (2-13C)-2,3-epoxytropone-
2-(13C)-carboxylate (18). Hence, these results suggest that
following the oxidation of substrate 16 by dehydrogenation,
TdaE surprisingly cleaves off the CoA moiety and epoxidizes
the tropone ring to afford the final product 18 (possibly via
intermediate 17), which decomposes to 9 by facile
decarboxylative epoxide ring opening during sample prepara-
tion and in the course of NMR and LCMS measurements
(Figures S27 and S32 and Figure 3C). Notably, in contrast to
previously reported similar epoxidation reactions,34 18
formation did not involve a 1,2-rearrangement of the
Product Characterization Reveals Subsequent TdaE-
Mediated CoA-Ester Cleavage and Ring Epoxidation.
The comparably low polarity of the newly formed TdaE
product suggested the loss of the CoA moiety, in line with the
results from LC-HRMS analysis (MH− m/z 165.018) that
pointed to the incorporation of another oxygen atom
−
1
(calculated for C8H5O4 m/z 165.019, Figure 3C, left
carboxylate group based on the JC,C doublet couplings
panel). In contrast, heterologously produced TdaD (Figures
S11, S12), a thioesterase-like enzyme previously speculated to
be responsible for CoA-ester cleavage in 11 biosynthesis and
also encoded in the Burkholderia spp. gene clusters (Figure 2),
processed neither 4 nor 16, which together with the
observation that TdaE itself eliminates CoA implies a different
function for TdaD. Both enzymes TdaEPi and TdaEBp catalyzed
the same reaction, suggesting that the conversion of 4 by TdaE
is relevant for the formation of tropolone as well as of sulfur-
containing tropone natural products (see Figure S13 and vide
observed for (13C2)-18 (Figure S29).
To determine the absolute configuration of 18, an
enantioselective synthesis of methyl (2S,3S)-2,3-epoxytro-
pone-2-carboxylate (24) was conducted starting from cyclo-
heptan-1,3-dione (19) by condensation with two units of
formaldehyde to 20 and reduction with diisobutylaluminum
hydride (DIBAL-H) to the allyl alcohol 21. Sharpless
epoxidation with L-(+)-diisopropyl tartrate to (2S,3S)-22
(94% ee), oxidation to the carboxylic acid 23, and methylation
resulted in (2S,3S)-24 (Figure 4A, for NMR spectra of
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Figure 4. Determination of the absolute configuration of enzymatically generated 18. (A) Enantioselective synthesis of (2S,3S)-24. Reaction
conditions: (a) paraformaldehyde, BF3·OEt2, CH2Cl2, room temperature, 3 h, 35%; (b) DIBAL-H, THF, −78 °C, 2 h, 78%; (c) L-(+)-diisopropyl
tartrate, Ti(OiPr)4, 4 Å molecular sieves, t-BuOOH, CH2Cl2, −17 °C, 20 h, 47%; (d) Jones reagent, acetone, room temperature, 3 h; (e)
trimethylsilyldiazomethane, Et2O, 0 °C, 40 min, 24% over two steps. (B) Microderivatization of 18: (f) Pd/C, MeOH, room temperature, 30 min;
(g) trimethylsilyldiazomethane, benzene, room temperature, 30 min.
Figure 5. Mechanistic scheme for TdaE catalysis. Note that a tautomer of 4 is shown as substrate for TdaE. The carbon numbering of all
compounds is according to Figure 1. See text for details on the individual steps. R = ribityl-ADP.
synthetic intermediates and 24, cf. Figures S33−S44). This
synthetic compound was compared to the TdaE product 18,
which was converted into 24 by microderivatization (catalytic
hydrogenation and methylation; Figure 4B). Analysis by GC/
MS on a chiral stationary phase in comparison to synthetic
(rac)-24 (prepared according to Scheme S3 in the Materials
and Methods, relevant NMR spectra are shown in Figures
S45−S53) and (2S,3S)-24 revealed that 24 obtained from the
enzyme product 18 is the opposite enantiomer of synthetic
(2S,3S)-24 (Figures S54) and thus has (2R,3R) configuration;
that is, the TdaE enzyme product is (2R,3R)-18.
TdaE Functions as an Archetypal Internal Flavopro-
tein Dioxygenase. To further study the formation of 18,
18O-isotope labeling experiments with H218O and 18O2 were
conducted. Unexpectedly, no 18O incorporation from H218O
into the carboxy group of 18 was observed by LCMS in the
TdaE assays, inconsistent with conventional hydrolytic CoA-
ester cleavage (Figures S55 and S56). Instead, two 18O atoms
were incorporated into 18 from 18O2 (Figure 3C), implying
that both added oxygens result from enzyme-mediated
oxygenation. This was further confirmed by the observed in-
source fragmentation of 18 via decarboxylation to 9 during the
LCMS measurements that demonstrated the loss of one of the
two 18O2-derived 18O labels (Figure 3C). Together, these data
suggest that TdaE functions as a rare internal oxygenase by
using its own substrate 4 as electron donor for flavin reduction
and O2 activation (rather than external oxygenases, which
require NAD(P)H as “co-substrate”).35 Consistent with that,
TdaE-Flox did not react with NAD(P)H based on UV−vis
spectroscopic analysis (Figure S57) and remained active in
enzyme assays lacking NADPH (normally required by
PaaABCE) that were started from purified 7 rather than 5
(Figure 3C, left panel; Figure S55). Notably, TdaE-Flox
reduction by 4 would likely require the C2-protonated
tautomer, which may be formed in the active site of TdaE.
In normal TdaE assays, however, such a tautomerization could
not be observed, most likely because the subsequent steps
proceeded too fast. To further investigate this, TdaE with low
FAD cofactor loading was used (to slow down dehydrogen-
ation), which indeed led to the rapid conversion of 4 into a
new compound that likely represents the proposed tautomer
(as shown in Figure 5). This compound also formed
spontaneously in much lower amounts in control samples
lacking TdaE (Figure 3, 9.5 min peak in the t(0 min) sample
and Figure S58).
Subsequent to 16 formation, TdaE-Flred reacts with O2 and
incorporates both activated oxygen atoms into the substrate,
which is highly unusual for flavoproteins that normally
exclusively function as monooxygenases.35 Moreover, the
chemical properties of the well-studied classical flavin-C4a-
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(hydro)peroxide (FlC4aOO(H)) oxygenating species35−37 seem
inconsistent with the observed reactions. First, the FlC4aOO(H)
species could only be formed once per catalytic cycle with two
available electrons for O2 activation, and despite many known
functions of FlC4aOO(H)-dependent FPMOs,35,38 the incorpo-
ration of both oxygens into a substrate has not been
reported.35,36 Second, the observed oxygenation chemistry
leads to Flox elimination via C2,C8-epoxide formation (steps V
and VI) and the generation of 18 (step VII).
TdaE Is Distinct from Classical ACAD-like Flavoen-
zymes. To analyze the relationship of TdaE with characterized
ACADs and group D FPMOs with ACAD fold, a multiple
sequence alignment and a homology model of TdaEPi was
generated. Strikingly, while TdaE operates as an oxygenase, the
predicted overall structure and active-site architecture more
resemble classical ACADs. Moreover, the sequence alignment
revealed highly conserved amino acids, including active site
residues, in all predicted functional homologues of TdaE from
both Burkholderiacea (β-Proteobacteria) and Roseobacteracea
(α-Proteobacteria). These residues were lacking in both
classical acyl-CoA dehydrogenases and group D FPMOs
(Figures S61 and S62), consistent with the unusual TdaE
functionality.
appears incompatible with the chemical properties of the
35−37
FlC4aOO(H)
,
specifically the required redox neutral transfer
of an [OH]− for CoA-ester oxygenolysis. Typically, such a
reaction is achieved by water-activating hydrolases such as
thioesterases. Recently, however, novel paradigms for FPMOs
were demonstrated that involved N5-oxygenated flavin
cofactors in the form of the flavin-N5-peroxide (FlN5OO) and
the flavin-N5-oxide (FlN5O) employed for the redox-neutral
oxygenolytic cleavage of carbon−heteroatom bonds (e.g., the
amide-bond cleaving pyrimidine oxygenase RutA)35,39 and for
polyketide hydroxylation (EncM from enterocin biosyn-
thesis),40,41 respectively. TdaE may thus constitute the first
member of a novel class of flavoprotein dioxygenases, which
combines FlN5OO and FlN5O catalysis by first oxygenolytically
cleaving the CoA ester with the FlN5OO species, before
epoxidizing the tropone ring with the help of the formed
FlN5O, thereby giving rise to 18 and Flox (Figures 1 and 5).
To gain a better understanding of the oxygenation
mechanism, it was further investigated whether TdaE-Flox
can convert its intermediate 16 in the absence of its native
electron donor 4. For that, 16 was isolated and then separately
incubated with TdaE. As anticipated, 16 was not processed by
TdaE in the presence of NAD(P)H, whereas formation of 18
was observed when Flred was generated by a separate flavin
reductase, once more underling that CoA-ester cleavage
proceeds oxygenolytically rather than by classical hydrolysis.
Importantly, in contrast to previous assays, 17 accumulated in
the presence of the flavin reductase aside from 18, which
suggests the partial reduction of FlN5O to Flox that prevented
the second oxygen transfer (Figure S59). This observation also
provides evidence that thioester oxygenolysis precedes ring
epoxidation, fully in line with the mechanistic proposal.
Crucially, scrupulous LC-HRMS analysis indicated small
amounts of transient FlN5O species in the TdaE assays
(quenched shortly after the reaction start before complete
conversion of 4 into 18) that could be identified based on
characteristic mass spectral data and retention time (Figure
S60). To test whether FlN5O catalysis proceeds via radical
intermediates, radical scavengers (ascorbate, 5,5-dimethyl-1-
pyrroline-N-oxide) were added to the enzyme assays. However,
5,5-dimethyl-1-pyrroline-N-oxide hardly affected catalysis, and
only mild effects were observed for ascorbate (≈30% lowered
product formation), pointing toward a nonradical epoxidation
mechanism.
TdaE Connects the Phenylacetate Catabolon with
Tropone Biosynthesis in Vivo. To further verify 18 as a key
intermediate in the biosynthesis of tropone natural products,
cell-free lysates from B. plantarii and P. inhibens were prepared
from liquid cultures in the production phases of 9 and 11,
respectively. The lysate from P. inhibens converted enzymati-
cally produced (13C8)-4 into (13C8)-18 (Figure S63), pointing
to the presence of TdaE during 11-production. This result was
confirmed by RT-qPCR, revealing a strong upregulation of
tdaEPi expression in the main phase of 11 production (Figure
S64, top). The requirement of TdaE for the generation of 11
was further shown by construction of a P. inhibens ΔtdaE
mutant in which the tdaE gene was replaced with a kanamycin
resistance cassette, leading to an abolished production of 11
under accumulation of 1 (Figure S65). Similar to that, the
production of 1 was previously reported in a 2-degrading
Azoarcus evansii mutant strain that lacked a functional 3-
oxidizing ALDH30 and therefore most likely accumulated 4
analogous to P. inhibens ΔtdaE. These observations suggest
that unprocessed 4 degrades to 1 within these mutant strains
by CoA-ester hydrolysis, decarboxylation, and oxidation.
Comparable to P. inhibens, (13C8)-4 was converted into
(13C8)-18 by the B. plantarii lysate, and strong upregulation
of tdaEBp expression was observed in the 9-production phase
(Figure S64, bottom). In addition, (13C8)-18 was more rapidly
transformed into (13C8)-9 by the cell lysate from B. plantarii in
comparison to that from P. inhibens or to the spontaneous
decomposition of 18 into 9 observed in the in vitro TdaE
assays (Figure S66). In the BGC of B. plantarii, a gene
encoding a putative decarboxylase (NCBI accession ID:
WP_052498255.1) was found in the vicinity of tdaEBp (Figure
2). To test if the corresponding enzyme boosts 9 formation, it
was heterologously produced with an N-terminal MBP-tag and
purified (Figure S67). However, the soluble decarboxylase-like
enzyme had no effect on the formation rate of key virulence
factor 9 in the in vitro enzyme assays, thus suggesting that
decarboxylation is possibly accelerated by another enzyme
(Figure S68). Overall, these data support the proposed role of
TdaE homologues as bacterial key enzymes for formation of
structurally diverse tropone-containing natural products by
sequestering shunt product 4 from the paa catabolon.
Taken together, TdaE catalysis may first involve the
deprotonation of the C3-hydroxyl group of 4 under
concomitant transfer of a C2-hydride to the N5 of FAD,
similar to oxidations catalyzed by classical ACADs (step I,
Figure 5). Then, the formed Flred reacts with O2 to the FlN5OO
(step II) most likely via transient flavin semiquinone (FlSQ)
and superoxide radicals.42 The CoA ester of the produced 16 is
subsequently attacked by the nucleophilic FlN5OO to form a
tetrahedral covalent adduct (step III), followed by CoA-ester
oxygenolysis via heterolytic cleavage of the peroxy species
(step IV). The resulting FlN5O should then be properly
positioned for a Michael addition at C8 of 17, which ultimately
DISCUSSION
■
In this work we provide evidence for a biosynthetic route in
which a dead-end product from aromatic catabolism is
sequestered by the bacterial flavoenzyme TdaE as precursor
for bioactive tropone natural products and thereby illustrate an
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unusual intertwining of primary and secondary metabolism.
Our genomic analyses revealed previously unknown BGCs
most likely required for 9 and 15 biosynthesis in B. plantarii
and B. cenocepacia, respectively, which encode homologues of
TdaE originally identified in the 11 BGCs of marine
Rhodobacteraceae(e.g., P. inhibens). The comparison and
investigation of both TdaEBp and TdaEPi showed that they
catalyze the virtual identical conversion of 4 into 18 via the
intermediates 16 and 17. TdaE homologues therefore appear
to play pivotal roles for the biosynthesis of an abundance of
structurally distinct tropone natural products including 9 as
well as more complex sulfur-containing 11, 14 (and other
roseobacticides B−K), and 15. This suggests that the final
TdaE product 18 represents an advanced intermediate for
formation of these compounds, which is supported by the
observed conversion of 4 into 18 by both cell-free lysates of
11-producing P. inhibens and 9-producing B. plantarii.
Remarkably, following the efficient TdaE-catalyzed conversion
of the catabolic shunt product 4 into 18, compound 9 is
spontaneously formed via rearomatization and decarboxyla-
tion, which is facilitated by the epoxide moiety of 18. Overall,
this suggests that TdaE is key to the formation of the critical
virulence factor 9 in B. plantarii and thus a driving factor for
rice seedling blight. Future studies may aim at the development
of TdaE inhibitors to shut down 9 formation in such
pathogens. On the other hand, the downstream biosynthetic
steps to sulfur-containing tropones are more elaborate,
requiring sulfur precursors presumably formed and incorpo-
rated into the tropone ring by PatB and TdaB homologues,
respectively (Figure 2).15,33 Notably, 18 seems predisposed to
react with nucleophiles, and sulfur incorporation may thus
proceed via 1,6-conjugate addition at C7 and epoxide ring
opening at C8 en route to 11. The biosynthesis of 15 and the
roseobacticides involves further steps such as the elimination
of the carboxyl side chain. Given the highly promising
pharmaceutical features and biotechnological potential of
these compounds that are also critical for numerous marine
and terrestrial symbiotic interactions,1 TdaE could therefore be
exploited for the future bioengineering of tropone natural
product producer strains.
The investigation of TdaE catalysis furthermore exposed an
unanticipated series of reactions. First, TdaE relies on its
substrate 4 as electron donor (rather than NAD(P)H) for O2
activation and covalent flavin-oxygen adduct formation. Then,
TdaE incorporates both O2-derived oxygen atoms into the
substrate most likely via transient FlN5OO and FlN5O species,
thereby breaking the CoA thioester bond and epoxidizing the
tropone ring. This novel paradigm for natural product tailoring
via N5-oxygenated flavins effectuating dioxygenation is
supported by the observed formation of the FlN5O species
during TdaE catalysis. Thus far, flavoproteins were exclusively
reported as monooxygenases that typically rely on transiently
produced FlC4aOO species to process a substantial variety of
different substrates.36−38,43−46 This is exemplified by the group
D FPMO p-hydroxyphenylacetate 3-hydroxylase,38,47 which
shares the ACAD protein fold with TdaE and utilizes the
canonical FlC4aOO species for aromatic hydroxylation.35,37 This
monooxygenase dogma hitherto also held true for flavoen-
zymes relying on N5-oxygenated flavin cofactors for catalysis,35
i.e., FlN5OO-dependent RutA-like group C FPMOs,39 which
generate the FlN5O as a byproduct (that is not used for a
second oxygen transfer),39,48 and the putative group I FPMO
EncM,40−42 which transiently forms the FlN5OO as a precursor
for its stable FlN5O oxygenating species.41,42 TdaE accordingly
represents the first known flavoprotein dioxygenase, and the
discovery of FlN5O(O) species in a third structural type of
flavoproteins furthermore underlines the notion that the
microenvironment around the flavin cofactor rather than the
overall fold controls O2 reactivity and thereby enzyme
functionality.
It is noteworthy that aminoperoxide species comparable to
the FlN5OO do not seem to play a role in organic chemistry
probably as a result of their instability.39 TdaE, however,
employs the FlN5OO for an unusual redox-neutral (non-
oxidative) oxygenation that involves the hydrolysis-like formal
transfer of an [OH]−, which is normally mediated by water-
activating enzymes rather than oxygenases. Accordingly, TdaE
constitutes, to the best of our knowledge, the first enzyme that
oxygenolytically rather than hydrolytically cleaves a CoA
thioester bond, analogous to the case of amide bond cleavage
by RutA.35,39,48,49 The FlN5OO is a potent soft α-nucleophile
that is distinct in reactivity from activated water (i.e., a hard
nucleophile).35,39 Hence, other RutA-like group C FPMOs
expectedly catalyze more demanding FlN5OO-dependent oxy-
genation reactions, as exemplified by C−Cl bond cleavage
(dehalogenation) of hexachlorobenzene by HcbA1 or C−S
bond cleavage of dibenzothiophene sulfone by DszA.35,39 Key
to such “pseudohydrolysis” reactions is the lone pair of
electrons of the flavin-N5, which enables the elimination of the
oxygenated product as a leaving group from a covalent FlN5OO
-
substrate adduct via heterolytic cleavage of the O−O
bond.35,39 This contrasts with classical oxidative oxygenation
chemistry by enzymes in which the cofactor serves as leaving
group and takes up the electrons during heterolytic peroxide
cleavage.35 However, while the FlN5O is seemingly a byproduct
of reactions catalyzed by RutA-like enzymes, the proficient
TdaE additionally utilizes this species for a second oxidative
oxygenation reaction via formal transfer of an [OH]+. The
FlN5O corresponds to a nitrone (an oxoammonium in the
resonance form) that can be converted to a nitroxyl radical
upon single-electron reduction. These functional groups are
widely used in synthetic chemistry also for radical and
nonradical oxidation and oxygenation reactions, e.g., the
nitroxyl radical PINO (phthalimide-N-oxyl) or the nitrone
TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl),35,50 and it is
therefore congruous that enzymes such as EncM and TdaE
evolved to exploit the FlN5O species. On the basis of the
electrophilic properties of 17 at the oxygenation site and
consistent with our results, we propose a Michael addition of
the nucleophilic FlN5O to the tropone ring that subsequently
enables epoxide formation under elimination of Flox, although a
radical mechanism cannot be ruled out.
CONCLUSION
■
In summary, TdaE, ostensibly an inconspicuous member of the
ACAD enzyme family, was revealed as a remarkably efficient
key tailoring enzyme for the biosynthesis of environmentally
and pharmaceutically important tropone natural products from
marine and terrestrial bacteria. TdaE catalysis combines
classical dehydrogenation with subsequent aminoperoxide
and aminoxide/nitrone chemistry to mediate CoA-ester
oxygenolysis and ring epoxidation via consecutive chemo-
and regioselective oxygen transfer steps. These findings
exemplify how a single enzyme can take advantage of the
distinct chemical features of two different oxygen transferring
species in the form of the FlN5OO and the FlN5O species to
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dioxygenase.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c04996.
■
sı
Experimental procedures and characterization data for
the reported compounds, Figures S1−S68, and Tables
S1−S4 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
■
Jeroen S. Dickschat − Kekulé-Institute of Organic Chemistry
and Biochemistry, University of Bonn, 53121 Bonn,
Germany; Institute of Organic Chemistry, TU Braunschweig,
38106 Braunschweig, Germany; orcid.org/0000-0002-
0102-0631; Email: dickschat@uni-bonn.de
Robin Teufel − Faculty of Biology, University of Freiburg,
79104 Freiburg, Germany; orcid.org/0000-0001-5863-
7248; Email: robin.teufel@zbsa.uni-freiburg.de
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and deoxygenates toxic epoxide. Nature 2012, 483, 359−362.
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Cygler, M. Structural and functional studies of the Escherichia coli
phenylacetyl-CoA monooxygenase complex. J. Biol. Chem. 2011, 286,
10735−10743.
(13) Spieker, M.; Saleem-Batcha, R.; Teufel, R. Structural and
Mechanistic Basis of an Oxepin-CoA Forming Isomerase in Bacterial
Primary and Secondary Metabolism. ACS Chem. Biol. 2019, 14,
2876−2886.
Authors
Ying Duan − Faculty of Biology, University of Freiburg, 79104
Freiburg, Germany
Marina Toplak − Faculty of Biology, University of Freiburg,
79104 Freiburg, Germany
Anwei Hou − Kekulé-Institute of Organic Chemistry and
Biochemistry, University of Bonn, 53121 Bonn, Germany
Nelson L. Brock − Institute of Organic Chemistry, TU
Braunschweig, 38106 Braunschweig, Germany
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inhibens. Chem. Commun. 2014, 50, 5487−5489.
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tropolone production and induction of a Burkholderia plantarii
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c04996
Author Contributions
⊥Y.D., M.T., and A.H. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Deutsche Forschungsgemein-
schaft (DFG) by grant nos. TE 931/3-1, TE 931/4-1 (awarded
to R.T.), and SFB-TR51 (“Roseobacter”) and by the Fonds zur
Förderung der Wissenschaftlichen Forschung (FWF) via an
Erwin-Schrödinger stipend (J4482-B) awarded to M.T. and a
Chinese Scholarship Council (CSC) grant 201606300019
awarded to Y.D.
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