Biosynthesis of the antibiotic tropodithietic acid by the marine bacterium Phaeobacter inhibens

Article abstract of DOI:10.1039/c4cc01924e

The biosynthesis of tropodithietic acid was investigated using a combinatorial approach of feeding experiments, gene knockouts and bioinformatic analyses. The mechanism of sulfur introduction is distinct from known mechanisms in holomycin, thiomarinol A a

Full text of DOI:10.1039/c4cc01924e

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Biosynthesis of the antibiotic tropodithietic acid
by the marine bacterium Phaeobacter inhibens†
Cite this: Chem. Commun., 2014,
50, 5487
Nelson L. Brock, Alexander Nikolay and Jeroen S. Dickschat*
Received 14th March 2014,
Accepted 3rd April 2014
DOI: 10.1039/c4cc01924e
www.rsc.org/chemcomm
The biosynthesis of tropodithietic acid was investigated using a combina- into a pathogen that promotes the collapse of the bloom.4 The bio-
torial approach of feeding experiments, gene knockouts and bioinformatic synthesis of 1–3 is largely unknown, but these tropone derivatives
analyses. The mechanism of sulfur introduction is distinct from known may have a common origin. A rare feature of 1 and 2 is the disulfide
mechanisms in holomycin, thiomarinol A and gliotoxin biosynthesis.
moiety that is also found in holomycin (4) from Streptomyces
clavuligerus5 and gliotoxin (5) from Aspergillus fumigatus.6
Feeding of 13C-labeled phenylalanine (Phe) and phenylacetic
Bacteria of the Roseobacter clade are associated with marine algae
that can appear in massive blooms.1 The bacteria are well known for acid (PAA) together with gene deletions showed that the carbon
their sulfur metabolism, especially for the degradation of the algal backbone of 1 arises via the PAA catabolon,7,8 in agreement with
metabolite dimethylsulfoniopropionate into dimethyl sulfide.2 Only labeling studies of its tautomer thiotropocin in Pseudomonas.9
a few secondary metabolites are known from the Roseobacter clade Transposon mutagenesis revealed several genes of the upper PAA
many of which contain sulfur. Structurally unique compounds from catabolon and of primary sulfur metabolism as well as a TDA
Phaeobacter inhibens include the antibiotics tropodithietic acid biosynthetic gene cluster (tdaA–tdaF, Fig. 2) to be relevant for TDA
(TDA, 1) and hydroxy-TDA (2),3 and the roseobacticide family of biosynthesis.7,10 TdaA is a positive regulator of the cluster,11
algicides, e.g. roseobacticide A (3, Fig. 1).4 In prosperous algal while the functions of the other encoded enzymes are unknown.
blooms 1 prevents bacterial infections, while lignin breakdown Here we report on the biosynthesis of 1 in P. inhibens.
products that are released in fading blooms upregulate roseobacti-
During PAA catabolism (Scheme 1A) 6 is converted into
cide biosynthesis. Upon this switch the bacterial symbiont turns phenylacetyl-CoA (7) by the CoA ligase PaaK. The aromatic ring
oxidation complex PaaABCDE converts 7 into the epoxide 8 that
is transformed into the oxepin 9 by the isomerase PaaG by a
protonation–deprotonation mechanism. The subsequently acting
PaaZ is a bifunctional enzyme with a C-terminal enoyl-CoA hydratase
(ECH) and a N-terminal aldehyde dehydrogenase (ALDH) domain.
The ECH domain catalyzes the water addition to 9 forming a hemi-
acetal that undergoes ring-opening and tautomerization to form 10.
This is followed by oxidation to the corresponding carboxylic acid
(11) by the ALDH domain. The next steps include a series of
b-oxidations to yield two units of acetyl-CoA and one succinyl-CoA.
A single mutation of a critical residue of E. coli PaaZ (E256Q) is
sufficient to switch the function of its N-terminal domain, resulting
in the conversion of 10 into 12 (Scheme 1B).8 In P. inhibens, besides
Fig. 1 Metabolites from P. inhibens (1–3), holomycin and gliotoxin.
paaZ1 of the PAA catabolon, a copy of this gene (paaZ2) is found
¨
Institut fur Organische Chemie, Hagenring 30, 38106 Braunschweig, Germany.
E-mail: j.dickschat@tu-braunschweig.de; Fax: +49 531 3915272;
Tel: +49 531 3915264
† Electronic supplementary information (ESI) available: Alignment of PaaZ
sequences, results from feeding experiments, experimental procedures, synthesis
of 34S-labeled amino acids, and NMR spectra of synthetic compounds. See DOI:
10.1039/c4cc01924e
Fig. 2 TDA biosynthetic gene cluster of P. inhibens DSM 17395.
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Scheme 3 Metabolism of sulfur amino acids.
Incorporation into both sulfur atoms of 1 was also observed from
[34S]-16 (87%) and [34S2]-17 (45%, this lower incorporation rate is
likely due to the poor water solubility of 17), demonstrating that
sulfur amino acid metabolism is involved in the biosynthesis of 1.
The incorporation of labeling from NaH34SO4 proceeds by
reduction to H34SÀ and reaction with O-acetyl serine (18) to form
[34S]-16 by the cysteine synthase CysK (Scheme 3). Incorporation
of Na234SSO3 was less efficient (30%) and is explained by oxidation
to sulfate via the Sox pathway.15 Transposon mutation3 or deletion
of patB encoding a cystathionine-b-lyase completely abolished
the production of 1 (Fig. S4 of ESI†) that was not restored by
cysteine or homocysteine addition. PatB also acts on 17 to yield
S-thiocysteine (21),16 thus pointing to 17 as a direct precursor
of 1. Incorporation of 34S-labeling was also observed in 2 in all
feeding experiments (Fig. S5 of ESI†).
A plausible mechanism for further elaboration of the carbon
backbone of 12 and sulfur introduction into 1 was deduced
from a bioinformatic analysis of the enzymes of the TDA gene
cluster (Table S1 of ESI†). Oxidation of 12 to 22 by the acyl-CoA
dehydrogenase TdaE and water elimination by the dehydratase
TdaC may yield the CoA ester 23 (Scheme 4). Intermediates 22
and 23 are reflected by the occurrence of tropone (25) and its
hydrate 2417 in headspace extracts of P. inhibens that arise by
thioester hydrolysis and decarboxylation.
The flavoprotein TdaF has a high homology to phosphopanto-
thenoylcysteine decarboxylases. These enzymes are involved in
coenzyme A biosynthesis and catalyze the flavine mononucleotide
(FMN) dependent oxidation of phosphopantothenoylcysteine (26)
to the thioaldehyde 27, followed by decarboxylation and reduction
with FMNH2, without net consumption of FMN (Scheme 5A).18
TdaB, homologous to glutathione-S-transferases, may catalyze
the addition of 21 to 23 that can be oxidized by TdaF and FMN
to yield 32 (Scheme 5B). The byproduct thioaldehyde 31 can
undergo decarboxylation and FMNH2 reduction to cysteamine,
similar to the respective steps in CoA biosynthesis. Addition of
Scheme 1 (A) PAA catabolon; (B) function of PaaZ-E256Q.
adjacent to tdaF (Fig. 2). While the C-terminal ECH domain of PaaZ2
is highly homologous to those of PaaZ1 and PaaZ of E. coli, the
N-terminal ALDH domain shows only weak sequence homology.
The highly conserved Cys-295, which covalently binds to the alde-
hyde function of 10 during catalysis and is critical for functionality,12
is mutated to Arg in PaaZ2, while a Cys residue aligns with Glu-256
of E. coli PaaZ (Fig. S1 and S2 of ESI†). Thus, PaaZ2 cannot catalyze
the conversion of 10 to 11, but serves in the formation of the seven-
membered carbocycle 12 to install the carbon backbone of 1.
To explore the formation of the dithiet moiety of 1 feeding
experiments with 34S-labeled compounds were performed.
Inorganic sulfur sources (Na234SSO3 and NaH34SO4) were obtained
as reported previously.13 For the synthesis of labeled cysteine (16)
34S8 was treated with a hot aqueous KCN solution to obtain
K34SCN (Scheme 2). Nucleophilic substitution at methyl N-(tert-
butoxycarbonyl)-O-tosyl-L-serinate (13) yielded the thiocyanate 14 that
was cleaved into the thiol 15 with SmI2, followed by acid deprotec-
tion to form [34S]-16. Its dimer [34S2]-17 was obtained by oxidation
with NaI and H2O2.14 Feeding of NaH34SO4 to P. inhibens, diluted to a
4% labeling due to the sulfate content in the medium, resulted in
the incorporation into both sulfur atoms of 1 with 4% incorporation
rate that was hence completely recovered (Fig. S3 of ESI†).
Scheme 2 Synthesis of 34S-labeled compounds.
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(Fig. S6 of ESI†), in full agreement with assumption (ii). Feeding
of 18O2 also resulted in the incorporation into one oxygen of the
shunt product 24 (Fig. S7 of ESI†), in agreement with the
pathways shown in Schemes 1 and 4.
We have investigated the biosynthesis of 1 and 2 by a com-
bined strategy of gene knockouts, bioinformatic analyzes, and
feeding experiments with labeled precursors. The delineated
pathway is in full agreement with all results from labeling,
mutation and complementation experiments, corroborated by the
presence of the shunt products tropone and tropone hydrate in
P. inhibens headspace extracts, and plausible in light of the
knowledge about characterized homologous enzymes. Particularly
interesting is the mechanism of sulfur introduction that is distinct
from recently unraveled mechanisms in holomycin, the related
thiomarinol A, and gliotoxin biosynthesis.19–21 Further studies on
the enzymes of TDA biosynthesis are now possible.
Scheme 4 Formation of the tropone ring and shunt products.
21 to the vinylogous Michael acceptor in 32 and oxidation can
introduce the second sulfur to yield 33 that may undergo
spontaneous oxidation to 34 in air. Finally, the thioesterase
TdaD catalyzes thioester hydrolysis to yield 1.
We also investigated the biosynthetic origin of the additional
oxygen in 2, assuming two alternatives: (i) formation of 1 from
tyrosine via p-hydroxy-PAA, and (ii) oxidation of 1 or a late stage
intermediate. Feeding experiments with [phenol-17O]tyrosine and
18O2 proceeded only with incorporation of labeling from 18O2
This work was funded by the DFG (SFB TR51 ‘‘Roseobacter’’)
and the Fonds der Chemischen Industrie with a Chemiefonds
Scholarship (to NLB).
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Scheme 5 (A) Mechanism of phosphopantothenoylcysteine decarboxyl-
ases; (B) introduction of sulfur into 1.
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Chem. Commun., 2014, 50, 5487--5489 | 5489