Bisucaberin biosynthesis: An adenylating domain of the BibC multi-enzyme catalyzes cyclodimerization of N-hydroxy-N-succinylcadaverine

Article abstract of DOI:10.1039/b813029a

The bisucaberin biosynthetic gene cluster has been identified in Vibrio salmonicida and a domain from within the BibC multienzyme encoded by the cluster has been shown to catalyse ATP-dependent dimerisation and macrocyclisation of N-hydroxy-N-succinylcadaverine to form bisucaberin. The Royal Society of Chemistry.

Full text of DOI:10.1039/b813029a

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Bisucaberin biosynthesis: an adenylating domain of the BibC multi-enzyme
catalyzes cyclodimerization of N-hydroxy-N-succinylcadaverinew
Nadia Kadi, Lijiang Song and Gregory L. Challis
Received (in Cambridge, UK) 29th July 2008, Accepted 5th September 2008
First published as an Advance Article on the web 22nd September 2008
DOI: 10.1039/b813029a
The bisucaberin biosynthetic gene cluster has been identified
in Vibrio salmonicida and a domain from within the BibC
multienzyme encoded by the cluster has been shown to
catalyse ATP-dependent dimerisation and macrocyclisation of
N-hydroxy-N-succinylcadaverine to form bisucaberin.
intermediates that are covalently attached to the multienzymes
throughout the assembly process.1
Bisucaberin 1 is a dimeric macrocylic metabolite of the
marine bacterium Alteromonas haloplanktis (Scheme 1).7 It
renders tumor cells susceptible to macrophage-mediated
cytolysis and also causes direct cytostasis by specific inhibition
of DNA synthesis.7 The macrocyclic bis-hydroxamate struc-
ture of bisucaberin is highly preorganized for ferric iron
binding; it has a much higher affinity for ferric iron than
acyclic bis-hydroxamate ligands.8 At low pH bisucaberin
predominantly forms a 1 : 1 complex with ferric iron, whereas
at neutral pH a monobridged 2 : 3 ligand : iron complex is the
predominant species.8 Bisucaberin may function as a side-
rophore in A. haloplanktis. However, this hypothesis remains
to be verified.
Macrocycles are key structural motifs of many bioactive natural
products, including clinically-used antibacterials, antifungals,
immunosuppressants and antiparasitics, as well as several iron-
chelating siderophores that play vital roles in bacterial iron
acquisition.1,2 Several macrocyclic natural products are
oligomeric and are composed of two or more identical or similar
subunits joined together by ester, amide, thioester or carbon–
carbon linkages.1–6 Recently, it has become apparent that the
key macrocyclization and oligomerization–macrocyclization re-
actions in the biosynthesis of many natural products are
catalyzed by thioesterase (TE) domains of nonribosomal peptide
synthetase (NRPS) and modular polyketide synthase
(PKS) multienzymes.1 These reactions proceed via a series of
Recently, bisucaberin has also been isolated from another
marine bacterium, Vibrio salmonicida, the causative agent of
cold-water vibriosis, a fatal septicaemia of farmed fish includ-
ing Atlantic salmon, cod and rainbow trout.9 Interestingly,
Scheme 1 Organization of the bib-bit and des gene clusters, and proposed roles of Bib and Des proteins in bisucaberin/desferrioxamine biosynthesis.
Genes encoding proteins with analogous functions have the same colour. The structures of fragment ions observed in the ESI-MS/MS spectrum of 5
are indicated.
another marine Vibrio species has been reported to produce
the acyclic tris-hydroxamate desferrioxamine G1 2.10 Bisuca-
berin 1 and desferrioxamine G1 2 both consist of alternating
succinic acid and N-hydroxycadaverine units linked by amide
bonds (Scheme 1). However, 1 contains two molecules each of
Department of Chemistry, University of Warwick, Coventry,
UK CV4 7AL. E-mail: g.l.challis@warwick.ac.uk;
Fax: 024 7652 4112;; Tel: 024 7657 4024
w Electronic supplementary information (ESI) available: Experimental
procedures, analysis of His6-BibCC expression and purification, spec-
troscopic and kinetic data. See DOI: 10.1039/b813029a
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succinic acid and N-hydroxycadaverine, whereas 2 contains
three molecules of each of these units.
We recently showed that the Streptomyces coelicolor DesD
enzyme catalyzes key trimerization and macrocyclization
reactions in desferrioxamine biosynthesis.2c DesD is the first
biochemically characterized member of a putative family of
novel synthetases that catalyze oligomerization–macrocycliza-
tion reactions via a different mechanism to the TE domains of
NRPSs and PKSs.2c It catalyzes ATP-dependent trimerization
of N-hydroxy-N-succinylcadaverine (HSC) 3 to form desfer-
rioxamine G1 2 and subsequent ATP-dependent macrocycli-
zation of 2 to form desferrioxamine E 4 (Scheme 1).2c We
speculated that bisucaberin 1 may be assembled by a DesD
homologue that catalyzes dimerization and subsequent
macrocyclization of HSC 3,2c rather than the trimerization–
macrocylization of HSC catalyzed by DesD in the biosynthesis
of desferrioxamine E 4.
Fig. 1 Extracted ion chromatograms (EICs) from LC-MS analyses of
the incubation of HSC 3 with His6-BibC, ATP and Mg2+. From top:
EIC at m/z = 401; EIC at m/z = 419; EIC at m/z = 401 from negative
control; EIC at m/z = 419 from negative control.
pNK003, which encodes a His6-BibCC fusion protein. The
plasmid was introduced into E. coli BL21star(DE3) and
His6-BibCC was overproduced as a soluble protein that was
purified to homogeneity from cell-free extracts of the expres-
sion host by Ni-NTA chromatography and subsequent gel
filtration. The gel formation indicated that His6-BibCC exists
as a dimer in solution. The identity of the purified protein was
confirmed by peptide mass fingerprinting.
Sequencing of the Vibrio salmonicida LFI1238 genome is
currently in progress at the Sanger Institute (http://www.sanger.
ac.uk/Projects/V_salmonicida/). We used the TBLASTN
algorithm to search the draft genome sequence of V. salmonicida
for DesD homologues.11 This analysis identified a putative gene,
which we named bibC (bisucaberin biosynthesis), encoding a 819
amino acid protein predicted to contain two functional
domains. The C-terminal domain (BibCC) shows 44% identity
and 63% similarity across a 603 amino acid overlap to DesD.2
The N-terminal domain (BibCN) shows 32% identity and 50%
similarity over a 172 amino acid overlap to DesC, which has
been proposed to catalyze the acylation of N-hydroxy-
cadaverine with succinyl-CoA to form HSC 3 during desferriox-
amine biosynthesis in Streptomyces species.2 This analysis sug-
gested that BibC is a multienzyme that catalyzes the formation
of HSC 3, followed by its dimerization and subsequent macro-
cyclization to form bisucaberin 1 (Scheme 1). We also analyzed
the coding sequences flanking bibC. Two putative genes (bibA
and bibB) upstream of bibC encode proteins that are similar to
the DesA and DesB enzymes, respectively, and that are pro-
posed to catalyze identical reactions in desferrioxamine and
bisucaberin biosynthesis (Scheme 1).2 Downstream of bibC we
identified five putative genes which we designated bitABCDE
(bisucaberin transport) that encode homologs of proteins
known to participate in the uptake of ferric-
siderophore complexes in Gram-negative bacteria (TonB-depen-
dent ferric-siderophore outer membrane receptor, putative fer-
ric-siderophore periplasmic binding protein, 2 Â permease
components and an ATPase component of ferric-siderophore
ABC transport systems, respectively) (Scheme 1).12 Taken
together these analyses suggest that the bibABC-bitABCDE
gene cluster encodes all the proteins required for the biosynthesis
of bisucaberin 1 and the uptake of its ferric complex(es). Intrigu-
ingly, two coding sequences flanking this gene cluster encode
transposase homologs, suggesting that it may have been acquired
by horizontal transfer into the genome of V. salmonicida.
To examine the involvement of the identified gene cluster in
bisucaberin biosynthesis and test the hypothesis that BibCC
catalyzes the dimerization and subsequent macrocyclization of
HSC 3, we cloned nucleotides 601-2460 of the bibC coding
sequence (encoding the C-terminal 619 amino acids of BibC
corresponding to the BibCC domain) into pET151 to create
Purified His6-BibCC was incubated with ATP, Mg2+ and
synthetic HSC 3.2c The reaction was stopped by addition of
trichloroacetic acid and LC-MS analysis identified two com-
pounds with retention times of 11.7 and 13.0 minutes and
m/z = 419 and 401, respectively (Fig. 1), which were absent
from control reactions containing His6-BibCC inactivated by
boiling. The compound with m/z = 401 was isolated from a
scaled-up incubation by semi-preparative HPLC. ESI-TOF-
+
MS analysis yielded the molecular formula C18H33N4O6 for
the m/z = 401 ion (calc.: 401.2395, found: 401.2397), consis-
tent with structure 1 for this compound. ESI-MS/MS of the
m/z = 401 ion produced a dominant fragment at m/z = 201,
as previously reported for bisucaberin isolated from V.
Salmonicida.9 The 1H NMR data for this compound were
identical to those reported for synthetic and natural bisuca-
berin.7,13 COSY, HSQC and HMBC experiments unambigu-
ously confirmed that the isolated compound was bisucaberin
1. Trace amounts of desferrioxamine E 4 were also observed
during these analyses.
The compound with m/z = 419 was isolated from a scaled-up
incubation that was stopped after 5 min. The molecular formula
+
of this ion was deduced to be C18H35N4O7 (calc.: 419.2500,
found: 419.2501) by ESI-TOF-MS analysis, consistent with
structure 5 for this compound. Although the compound could
not be isolated in sufficient amounts to characterize by NMR
spectroscopy (because it is a transient intermediate in the
conversion of 3 to 1—see below), ESI-MS/MS analysis
generated daughter ions with m/z = 319 and 201 from the
m/z = 419 [M + H]+ ion, clearly indicating that it is
structurally related to 1 and providing strong evidence for
structure 5 (Scheme 1).
To investigate whether 5 is an intermediate in the conversion
of HSC 3 to biscucaberin 1, we incubated HSC with purified
His6-BibCC, Mg2+ and ATP for 2.5, 5, 7.5, 10, 20, 30, 45 and
60 min. The reactions were halted by addition of ferric chloride,
which precipitated the enzyme and converted 1 and 5 to their
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domain within a multienzyme, providing an unexpected
parallel with nonribosomal peptide and polyketide biosynth-
esis, where multienzymes are also involved.1 BLAST searches
indicate that similar multienzymes to BibC are encoded by
other NIS gene clusters in the database (see ESIw). Intrigu-
ingly, the DesD enzyme catalyzes selective trimerisation and
subsequent macrocyclization of HSC 3 in desferrioxamine
biosynthesis,2a whereas the BibCC domain catalyzes selective
dimerization and subsequent macrocyclization of HSC to
Scheme 2 Conversion of ATP to AMP and PPi in the BibCC-catalyzed
assembly of bisucaberin 1 from HSC 3 (A = adenosine).
corresponding ferric complexes. The formation of the ferric
complexes was confirmed by LC-DAD-MS; the complexes
exhibited absorbance maxima at 471 nm and produced ions
with m/z 454.3 and 472.3 in positive ion mode, consistent with
the expected formation of 1 : 1 iron : ligand complexes.8 The
relative quantity of the ferric complexes of 1 and 5 in each
incubation mixture was determined using HPLC monitoring
absorbance at 471 nm. A plot of the concentration of each
complex against time indicated that 5 is a transient intermediate
in the conversion of HSC 3 to bisucaberin 1 (see ESIw).
Compound 5 was unambiguously confirmed as an intermediate
in the conversion of HSC 3 to bisucaberin 1 by incubating a
purified sample of it with purified His6-BibCC, Mg2+ and ATP.
LC-MS analyses of the ferric complexes that resulted from
halting the reaction by addition of ferric chloride indicated that
5 had been completely converted to 1. No conversion was
observed in a control reaction using heat-inactivated enzyme.
Using coupled continuous assays for AMP and ADP, as
well as for phosphate and pyrophosphate (PPi), we showed
that BibCC converts ATP to AMP and PPi (Scheme 2 and
ESIw), consistent with the formation of acyl adenylate inter-
mediates during the assembly of bisucaberin 1 from HSC 3.
Taken together, our data lead us to propose a mechanism for
BibCC-catalyzed biosynthesis of bisucaberin 1 from HSC 3, as
follows. A molecule of ATP and a molecule of HSC 3 bind to
the active site of BibCC and react to form an adenylate. A
second molecule of HSC binds to the active site and undergoes
general base-mediated condensation with the adenylate to form
5, which can dissociate from the active site. The PPi and AMP
formed by the adenylation and condensation reactions dissoci-
ate from the active site and are replaced by a second molecule of
ATP. This reacts with bound 5 to form an adenylate that
undergoes macrocyclization via general base-mediated intramo-
lecular condensation of the amino group and the activated
carboxyl group in the adenylate to form 1, which is released
along with a molecule of AMP and a molecule of PPi from the
active site of BibCC. Alternative orders of substrate binding and
intermediate/product release are also possible. Further experi-
ments will be required to discriminate between these.
form bisucaberin 1. Both enzymes employ
a catalytic
mechanism for oligomerization and macrocyclization that
involves free intermediates. This mechanism is very different
from the mechanism of the oligomerization and macrocycliza-
tion reactions catalyzed by thioesterase domains of NRPSs
and PKSs, where all intermediates in the process remain
covalently bound to the enzyme throughout.1 The DesD
enzyme and the BibCC domain share 66% sequence similarity,
raising the intriguing question: how do two such similar
enzymes control the selective formation of trimeric and di-
meric macrocycles, respectively, from a common intermediate?
This work was supported by a grant from the UK BBSRC
(Grant ref: BB/S/B14450). We thank Prof. Nils-Peder
Willassen for providing Vibrio salmonicida LFI1238 genomic
DNA.
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Chem. Commun., 2008, 5119–5121 | 5121