Angewandte
Chemie
DOI: 10.1002/anie.201307406
Polyketide Biosynthesis Very Important Paper
An Enzymatic Domain for the Formation of Cyclic Ethers in Complex
Polyketides**
Petra Pçplau, Sarah Frank, Brandon I. Morinaka, and Jçrn Piel*
Five- and six-membered cyclic ether moieties are structural
features of many bioactive natural products. Examples are the
bacteria- and sponge-derived polyketides shown in Figure 1A
as well as many dinoflagellate toxins. Since such heterocycles
can be important pharmacological determinants, much effort
has been invested in understanding their formation in
to DH domains, but features aberrant active-site motifs
(Figure S1) and falls into a distinct phylogenetic clade (Fig-
[
6]
ure S2). This PKS component, provisionally termed pyran
[
6c]
synthase (PS) domain,
closure via oxa-conjugate cyclization as part of the reaction
was proposed to catalyze ring
[6a,c]
sequence shown in Figure 1B.
All known modules of this
[1]
[2]
nature and by stereoselective synthesis. Considerable
type feature the same overall architecture, suggesting a gen-
eral biosynthetic strategy used by PKSs to install diverse
cyclic moieties exhibiting different ring sizes, stereochemistry,
and substitution patterns.
[
3]
biosynthetic detail is known for polyether ionophores that
are matured by means of intriguing cyclization cascades
catalyzed by free-standing epoxide hydrolases. However, as
these enzymes act on complex substrates at the post-
polyketide synthase (PKS) stage, their development as more
general tools in chemoenzymatic synthesis might be challeng-
ing.
To investigate the function of this domain, we expressed
the PS of the biosynthetic pathway of pederin (1) from an as-
yet unculturable symbiotic Pseudomonas sp. bacterium asso-
[
6a,7]
ciated with Paederus spp. rove beetles.
Pederin (1) is
Many polyketide shown (1–4) or suspected (e.g., 5) to be
generated by a PKS group termed trans-acyltransferase PKS
a potent cytotoxin and blistering agent that is used by these
[
8]
beetles as chemical defense. The PS was hypothesized to
participate in the generation of the eastern tetrahydropyran
(THP) moiety with an anti substitution pattern at the ether
[
4]
(trans-AT PKS) contain ether rings that are likely intro-
duced during chain elongation. trans-AT PKSs are giant
modular enzymes, in which each module typically elongates
the growing polyketide chain by one building block. The
minimal domain set is a ketosynthase (KS) for chain
elongation and an acyl carrier protein (ACP), to which acyl
intermediates are attached by a thioester bond. Optional
further processing at this stage is controlled by additional
domains that may be present on the module. An important
characteristic of trans-AT PKSs is the great diversity of
module variants as a result of unusual domains and domain
[
6a]
a positions.
Domain boundaries were determined by
alignment to other homologues, including DH domains
(Figure S1). After amplification by polymerase chain reaction
(PCR) and cloning into pET29a, the PS was expressed and
purified as a C-terminally His -tagged protein (Figures S3–
6
S5). As test substrates, we synthesized thioesters 6 and 7 along
with standards for potential products 8 and 9, using the routes
shown in Scheme 1. These compounds harbor N-acetylcyste-
amine thioester (SNAC) units as simplified surrogates of the
ACP-bound 4’-phosphopantetheinyl thioesters in polyketide
biosynthesis. The E-configured double bond of the natural
substrate was assumed on the basis of the stereospecificity
motif of the KR located within the same module, which
usually correlates with the olefin configuration after dehy-
[
4,5]
combinations.
In addition to the canonical ketoreductase
(
KR), dehydratase (DH), and enoylreductase (ER) domains
that also occur in other type I polyketide and fatty acid
synthase systems, a wide range of further domains are found,
many of which have poorly understood functions. For trans-
AT PKS modules that introduce building blocks attached to
cyclic ethers, a characteristic feature is the presence of
a unique domain that exhibits moderate protein homology
[
9]
dration. HPLC (Figure 2) and NMR analyses showed that
the conjugated thioesters undergo negligible spontaneous
ring closure.
Enzyme assays were performed by incubating 6 and 7 in
separate reactions with the expressed PS and monitored by
HPLC. New product peaks were detected for both test
reactions, but not in controls containing boiled enzyme
(Figure 2), indicating conversion of the substrates. For test
substrate 6 with a primary alcohol function, HPLC profiles
exhibited a single additional peak (Figure 2A), while for the
secondary alcohol 7, two new peaks (peaks I and II in
Figure 2B) were observed. High-resolution MS analysis
suggested that all new peaks belong to compounds with
molecular formulae identical to that of the respective test
substrate (for the product of 6: m/z = 246.1158, calcd
246.1158, corresponding to C H NO S; products of 7:
[
*] P. Pçplau, S. Frank, Prof. Dr. J. Piel
Kekulꢀ Institute of Organic Chemistry and Biochemistry
University of Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
Dr. B. I. Morinaka, Prof. Dr. J. Piel
Institute of Microbiology
Eigençssische Technische Hochschule (ETH) Zurich
Wolfgang-Pauli-Strasse 10, 8093 Zurich (Switzerland)
E-mail: jpiel@ethz.ch
[
**] We thank Roy Meoded for technical assistance with protein
expression. We are grateful for financial support from the EU
(
BlueGenics to J.P.), the DFG (SFB 642 and PI 430/8-1 to J.P.), and
the Alexander von Humboldt Foundation (to B.M.).
1
1
20
3
m/z = 282.1139 and 282.1144, calcd 282.1140, corresponding
to C H NO SNa). The UV spectra of all products had
1
2
21
3
Angew. Chem. Int. Ed. 2013, 52, 13215 –13218
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
13215
Poeplau, Petra
