Unusual acetylation-elimination in the formation of tetronate antibiotics

Article abstract of DOI:10.1002/anie.201301680

The identity and reactivity of the intermediates in agglomerin biosynthesis were established and the respective roles of the acetyltransferase Agg4 and the eliminating enzyme Agg5 identified (see scheme). It is proposed that enzymes homologous to Agg4 and

Full text of DOI:10.1002/anie.201301680

Angewandte
Chemie
DOI: 10.1002/anie.201301680
Tetronate Antibiotics
Unusual Acetylation–Elimination in the Formation of Tetronate
Antibiotics**
Chompoonik Kanchanabanca, Weixin Tao, Hui Hong, Yajing Liu, Frank Hahn,
Markiyan Samborskyy, Zixin Deng, Yuhui Sun,* and Peter F. Leadlay*
À
Tetronate antibiotics comprise an important and growing
family of polyketide natural products possessing a character-
istic tetronate (4-hydroxy-[5H]furan-2-one) ring system. They
have been isolated from both terrestrial and marine bacteria,
and show a diverse range of biological activities.[1] They
include the tetronate polyethers tetronomycin[2] and tetrona-
sin,[3] the fatty acyltetronate antibiotic agglomerin,[4] and the
structurally closely related protein phosphatase inhibitor RK-
682[5] (Figure 1). Of particular interest are the structurally
intriguing spirotetronates (Figure 1a), including the antibac-
terial compounds chlorothricin[6] and abyssomicin,[7] the
antiviral compound quartromicin,[8] and the antitumour
compounds tetrocarcin[9] and kijanimicin.[10] These com-
pounds appear to arise through an enzyme-catalyzed Diels–
Alder reaction[11] after specific dehydration of an initially
formed tetronate precursor, as shown for atrop-abyssomi-
cin C[7] in Figure 1b.
that might catalyze formation of the tetronate ring C C and
À
C O bonds. Reconstitution of RK-682 biosynthesis in vitro
has been used to show that RkE is a glyceryl-S-acyl carrier
protein (ACP) synthase,[12] and that the ketoacyl-S-ACP
synthase FabH-like RkD is necessary and sufficient to
catalyze formation of the tetronate ring in vitro starting
from a 3-ketoacyl thioester and glyceryl-S-ACP.[5] Similar
results were recently obtained for the FabH-like QmnD5 in
quartromicin biosynthesis[8] and it seems highly likely that
most (if not all)[13] tetronates follow an analogous biosynthetic
pathway.
Until now, the course of the dehydration step in spiro-
tetronate biosynthesis, which provides the dienophile for the
ensuing Diels–Alder-like reaction, has remained obscure. We
show here, by cloning and analysis of the gene cluster for
biosynthesis of agglomerins A–D in Pantoea agglomerans
(formerly Enterobacter agglomerans) PB-6042,[4] its heterol-
ogous expression in Escherichia coli, and the total reconsti-
tution of agglomerin biosynthesis in vitro, that the mechanism
of dehydration actually involves two steps after formation of
the tetronate ring: O-acetylation catalyzed by Agg4, followed
by elimination of acetic acid to form the exocyclic double
bond catalyzed by Agg5. We propose that the biosynthesis of
spirotetronates involves the same two-step reaction sequence,
catalyzed by enzymes homologous to Agg4 and Agg5.
The agglomerin biosynthetic pathway of P. agglomerans
provided an attractive system in which to study these key
steps, given the relative simplicity of the structures of
agglomerin A and its congeners agglomerins B–D, which
differ from each other only in the nature of the fatty acyl
sidechain (Figure 1).[4] Cosmid and whole-genome sequencing
of P. agglomerans PB-6042 was used to reveal a circular
chromosome of approximately 4.3 Mbp (bp = base pairs),
within which the agglomerin cluster was readily identified
through its similarity to that of RK-682 (Supporting Informa-
tion, Figure S1).
Analysis of the biosynthetic gene clusters for several of
these natural products[3,5–10] has highlighted the presence of
a set of highly conserved genes unique to tetronate biosyn-
thesis, whose predicted products include candidate enzymes
[*] C. Kanchanabanca,[+] Dr. H. Hong, Dr. M. Samborskyy,
Prof. Dr. P. F. Leadlay
Department of Biochemistry, University of Cambridge
80 Tennis Court Road, Cambridge CB2 1GA (UK)
E-mail: pfl10@cam.ac.uk
Dr. W. Tao,[+] Y. Liu, Prof. Dr. Z. Deng, Prof. Dr. Y. Sun
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery
(Wuhan University), Ministry of Education and School of Pharma-
ceutical Sciences, Wuhan University
185 East Lake Road, Wuchan 430071 (P.R.China)
E-mail: yhsun@whu.edu.cn
Dr. F. Hahn
Institute of Organic Chemistry, Leibniz Universitꢀt Hannover
Callinstrasse 22, 30167 Hannover (Germany)
[+] These authors contributed equally to this work.
A 12 kbp DNA sequence encodes seven ORFs that could
be plausibly assigned to the cluster. As well as the expected
high homology between several genes in the agg and rk
clusters, key differences in enzymology could also be inferred
from the comparison of these clusters: whereas RK-682
obtains its linear precursor from palmitic acid, which is
activated and then elongated on a modular polyketide
synthase (PKS) to give 3-oxo-stearoyl-S-ACP,[12] no counter-
part of the rkC PKS could be found anywhere on the P.
agglomerans chromosome. The precursors for agglomer-
ins A–D appear to be taken directly from primary metabo-
lism, probably as the corresponding 3-oxoacyl-CoA thio-
esters. In support of this, when the seven genes of the putative
[**] This research was supported by grants from the Biotechnology and
Biological Sciences Research Council (BBSRC BB/D018943/1), and
the 973 and 863 programs from the Ministry of Science and
Technology, the National Natural Science Foundation of China, the
Ministry of Education. C.K. acknowledges a stipend from the Higher
Education Commission of Thailand, W.T. acknowledges funding
from the China Postdoctoral Science Foundation (CPSF
2012M521461), and F.H. funding as an EU Marie Curie Postdoc-
toral Fellow. F.H. is an Emmy Noether Fellow of the Deutsche
Forschungsgemeinschaft, and P.F.L. is a Research Awardee of the
Alexander von Humboldt Foundation. We thank Prof. Dr. R. D.
Sꢁssmuth and Dr. F. Huang for helpful discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301680.
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Figure 1. a) The structures of agglomerins, RK-682, tetronomycin and of representative spirotetronate antibiotics whose gene cluster has been
identified. b) Proposed common mechanism of spirotetronate biosynthesis exemplified for abyssomicin (adapted from Ref. [7]).
agg cluster were cloned into E. coli, the resulting recombinant
strain produced agglomerins A–D (Supporting Information).
Strikingly, the agg cluster does contain two adjacent genes
(agg4 and agg5) not found in the rk cluster, but which have
counterparts in all characterized spirotetronate gene clusters
(Supporting Information). We therefore particularly wished
to define the contribution of these genes to agglomerin
biosynthesis.
First, we individually expressed in E. coli each of Agg1
(FabH-like ketosynthase), Agg2 (glyceryl-S-ACP synthase),
Agg3 (ACP), Agg4, and Agg5. All five were obtained as
purified recombinant proteins, although the yields of Agg4
2
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and Agg5 were significantly lower than those of the other
proteins (Supporting Information). We have previously
reported[2] that the Agg4 homologue in tetronomycin biosyn-
thesis, Tmn7, has significant sequence similarity to the C-
terminal catalytic domain of the E2 (acyltransferase) compo-
nent of 2-oxoacid dehydrogenase multienzymes,[14] and that
all the conserved acyltransferase active-site residues are
present in Tmn7. The E2 domain has a known tertiary and
quaternary structure that closely resembles that of acetyl-
CoA:chloramphenicol acetyltransferase,[14] making it an
attractive idea that acetyl-CoA might be a co-substrate for
the enzymatic activity of Agg4 as well.
To test this idea, glyceryl-S-Agg3 (1; Scheme 1) was
produced in vitro as previously described[5,12] by generating
the glycolytic intermediate 1,3-bisphosphoglycerate in situ in
the presence of Agg2 and the acylcarrier protein Agg3
(Supporting Information). The other partner needed for
tetronate formation to form “hydroxy-agglomerin A” (3) was
predicted to be 3-keto-dodecanoyl-CoA (2). This was synthe-
sized in six steps starting from decanoic acid (Supporting
Information). When glyceryl-S-Agg3 was incubated with 2 in
the presence of purified Agg1, it led to the formation of
a species whose molecular mass, as judged by HPLC-MS
(Figure 2 and Supporting Information), exactly matched that
predicted for hydroxy-agglomerin A (3-decanoyl-5-hydroxy-
methyl-tetronate; 3; Scheme 1). This result supports the view
that 2 is the natural substrate for the ring-forming reaction.
When purified 3 was incubated with both Agg4 and
acetyl-CoA, a species was produced with a molecular mass
(Figure 2 and Supporting Information) exactly matching that
predicted for acetyl-agglomerin A (3-decanoyl-5-acetoxy-
methyl-tetronate; 4). No reaction occurred if either Agg4 or
acetyl-CoA were omitted from the incubation. Likewise, no
reaction occurred when Agg5 alone was incubated with 3. In
contrast, when acetyl-CoA, Agg4 and Agg5 were all present,
the product of the reaction was a species with a molecular
mass that exactly matched that of authentic agglomerin A
(Figure 2). When [2H3]-acetyl-CoAwas used instead of acetyl-
CoA in the incubation with Agg4, the observed molecular
mass of the putative acetyl-agglomerin A (4) was shifted
higher by three mass units (Supporting Information), as
expected. The high resolution masses of 3 (calculated:
283.1551; found: 283.1549) and 4 (calculated: 325.1657;
found 325.1657), as well as their MS2 fragmentation patterns
(Supporting Information), were in full agreement with the
structures assigned to them. These results taken together
support a two-step mechanism (Scheme 1) for formation of
the exocyclic double bond in agglomerins: first, Agg4
catalyzes the acetylation of the primary hydroxy group at
C-6 of pre-agglomerin; and Agg5 then catalyzes proton
abstraction from C-4 and concomitant departure of acetate as
a good leaving group. This mirrors the well-established
chemical transformation of RK-682 and its analogs into
their agglomerin counterparts through mesylation and sub-
sequent base-catalyzed elimination.[15] To our knowledge, this
mechanism for elimination has no direct enzymatic prece-
dent, but it is strongly reminiscent of the phosphorylation–
elimination sequence that is invoked for the formation of
dehydro-Ser and dehydro-Thr residues during lantibiotic
biosynthesis.[16,17]
We next wished to establish whether the proposed
mechanism for formation of the exocyclic double bond is
likely to operate in vivo. To address this, we developed
a system for carrying out specific gene deletion in P.
agglomerans; and used it to create mutant strains specifically
lacking agg4 and/or agg5 (see Supporting Information).
Fermentation of the mutants was carried out under conditions
where agglomerins A–D are produced by wild-type P.
agglomerans, and ethyl acetate extracts of the fermentation
media were analyzed by HPLC-MS. The analysis showed that
under these conditions the Dagg4 and Dagg4Dagg5::cat
strains produced only the respective hydroxy-agglomer-
ins A–D, while the Dagg5::cat strain produced as major
metabolites the respective acetyl-agglomerins A–D (Support-
ing Information), consistent with the intermediacy of these
species in agglomerin biosynthesis and with the roles ascribed
to Agg4 and Agg5.
When a mixture of hydroxy-agglomerins A–D, purified
from the Dagg4 strain, was incubated in vitro with purified
Agg4 and acetyl-CoA, all were converted to the respective
acetyl-agglomerins. When Agg5 was also included, the
product mixture was identical in HPLC-MS and HPLC-MS2
Scheme 1. Proposed pathway for tetronate formation and dehydration
in agglomerin biosynthesis. Intermediate 2 is likely to be derived from
fatty acid biosynthesis.
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chemoenzymatic syn-
thesis of authentic sub-
strates that can be used
to probe directly the
mechanism of the sub-
sequent Diels–Alder
rearrangement in spi-
rotetronate biosynthe-
sis, the catalyst for
which
remains
unknown.
Received: February 26,
2013
Published online: &&
&&, &&&&
Keywords: antibiotics ·
.
biosynthesis ·
dehydration ·
polyketide · tetronate
[1] R. Schobert, Natur-
wissenschaften 2007,
94, 1 – 11.
Figure 2. HPLC-MS analysis of tetronate intermediates in agglomerin biosynthesis in vitro: a) 3 formed in the
presence of Agg1, glyceryl-S-Agg3 and 3-keto-dodecanoyl-CoA in 50 mm Tris-HCl, pH 7.2; b) 4 formed from 3 in the
presence of Agg4 and acetyl-CoA; c) agglomerin A formed from 3 in the presence of Agg4, Agg5, and acetyl-CoA.
[2] Y. Demydchuk, Y.
Sun, H. Hong, J.
Staunton,
Spencer, P. F. Leadlay, ChemBioChem 2008, 9, 1136 – 1145.
[3] D. H. Davies, E. W. Snape, P. J. Suter, T. J. King, C. P. Falshaw, J.
Chem. Soc. Chem. Commun. 1981, 1073 – 1074.
J. B.
behavior with the respective agglomerins (Supporting Infor-
mation).
We reasoned that Agg4 and Agg5 might be sufficiently
versatile to accept RK-682 (3-palmitoyl-5-hydroxymethyl-
tetronate) as an alternative substrate to give a novel agglom-
erin. When RK-682 was incubated with purified Agg4 and
acetyl-CoA, a species was formed with the expected mass of
acetyl-RK-682 (calculated 409.2596; found 409.2598); and
inclusion of Agg5 in the incubation led to the appearance of
a new species with the expected mass of dehydro-RK-682 (3-
palmitoyl-5-methylidene-tetronate), the predicted novel
agglomerin with the fatty acyl chain characteristic of RK-
682 (calculated 349.2384; found 349.2382). The products of
incubation of Agg4 and acetyl-CoA with RK-682 were also
analyzed by 1H NMR spectroscopy (Supporting Information).
The deduced structure was in full accord with our proposed
mechanism. In particular, the acetyl group in the acetyl-RK-
682 is found to be attached via the C-6 hydroxy, as expected.
Our present results establish both the identity and the
chemical competence of the intermediates in agglomerin
biosynthesis shown in Scheme 1, and identify the respective
roles of the acetyltransferase Agg4 and the eliminating
enzyme Agg5. Although the predicted protein fold of
Agg5[17] places it within the a/b hydrolase superfamily, it has
no significant sequence similarity to proteins of known
function apart from homologues in known or suspected
tetronate biosynthetic pathways. We propose that enzymes
homologous to Agg4 and Agg5 carry out the dehydration
steps in all spirotetronate biosynthetic pathways. If this proves
correct, it may assist knowledge-based engineering of these
pathways to create novel analogues. Our identification of the
role of these novel enzymes should also now allow facile
[4] a) Y. Terui, R. Sakazaki, J. Shoji, J. Antibiot. 1990, 43, 1245 –
1253; b) Y. Mashimo, Y. Sekiyama, H. Araya, Y. Fujimoto,
Bioorg. Med. Chem. Lett. 2004, 14, 649 – 651.
[5] Y. Sun, F. Hahn, Y. Demydchuk, J. Chettle, M. Tosin, H. Osada,
P. F. Leadlay, Nat. Chem. Biol. 2010, 6, 99 – 101.
[6] X. Y. Jia, Z.-H. Tian, L. Shao, X.-D. Qu, Q.-F. Zhao, J. Tang, G.-
L. Tang, W. Liu, Chem. Biol. 2006, 13, 575 – 585.
[7] E. M. Gottardi, J. M. Krawczyk, H. von Suchodoletz, S. Schadt,
A. Mꢀhlenweg, G. C. Uguru, S. Pelzer, H. P. Fiedler, M. J. Bibb,
J. E. Stach, R. D. Sꢀssmuth, ChemBioChem 2011, 12, 1401 –
1410.
[8] H.-Y. He, H.-X. Pan, L.-F. Wu, B.-B. Zhang, H.-B. Chai, W. Liu,
G.-L. Tang, Chem. Biol. 2012, 19, 1313 – 1323.
[9] J. Fang, Y. Zhang, L. Huang, X. Jia, Q. Zhang, X. Zhang, G.-L.
Tang, W. Liu, J. Bacteriol. 2008, 190, 6014 – 6025.
[10] H. Zhang, J. A. White-Phillip, C. E. MelanÅon 3rd, H.-J. Kwon,
W. L. Wu, H.-W. Liu, J. Am. Chem. Soc. 2007, 129, 14670 – 14683.
[11] C. A. Townsend, ChemBioChem 2011, 12, 2267 – 2269.
[12] Y. Sun, H. Hong, F. Gillies, J. B. Spencer, P. F. Leadlay,
ChemBioChem 2008, 9, 150 – 156.
[13] Y. Sekiyama, Y. Fujimoto, K. Hasumi, A. Endo, J. Org. Chem.
2001, 66, 5649 – 5654.
[14] A. Mattevi, G. Obmolova, K. H. Kalk, A. H. Westphal, A.
de Kok, W. G. Hol, J. Mol. Biol. 1993, 230, 1183 – 1199.
[15] R. Schobert, C. Jagusch, J. Org. Chem. 2005, 70, 6129 – 6132.
[16] C. Chatterjee, L. M. Miller, Y. L. Leung, L. Xie, M. Yi, N. L.
Kelleher, W. A. van der Donk, J. Am. Chem. Soc. 2005, 127,
15332 – 15333.
[17] B. Krawczyk, P. Ensle, W. M. Mꢀller, R. D. Sꢀssmuth, J. Am.
Chem. Soc. 2012, 134, 9922 – 9925.
[18] L. A. Kelley, M. J. E. Sternberg, Nat. Protoc. 2009, 4, 363 – 371.
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Communications
Tetronate Antibiotics
C. Kanchanabanca, W. Tao, H. Hong,
Y. Liu, F. Hahn, M. Samborskyy, Z. Deng,
Y. Sun,* P. F. Leadlay*
&&&& — &&&&
Unusual Acetylation–Elimination in the
Formation of Tetronate Antibiotics
The identity and reactivity of the inter-
mediates in agglomerin biosynthesis
were established and the respective roles
of the acetyltransferase Agg4 and the
eliminating enzyme Agg5 identified (see
scheme). It is proposed that enzymes
homologous to Agg4 and Agg5 carry out
the dehydration steps in all spirotetronate
biosynthetic pathways. If this proves
correct, it may assist engineering of these
pathways.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!