Macrodiolide formation by the thioesterase of a modular polyketide synthase

Article abstract of DOI:10.1002/anie.201500401

Elaiophylin is an unusual C₂-symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C₂-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides. Think again: The thioesterase/cyclase enzyme of the elaiophylin polyketide synthase catalyzes symmetric diolide formation in vitro from a synthetic pentaketide substrate by an iterative mechanism. Unexpectedly, a tetraketide that is not itself a substrate can be co-opted in the presence of the pentaketide to produce an asymmetric macrodiolide.

Full text of DOI:10.1002/anie.201500401

Angewandte
Chemie
DOI: 10.1002/anie.201500401
Biosynthesis
Macrodiolide Formation by the Thioesterase of a Modular Polyketide
Synthase**
Yongjun Zhou, Patrꢀcia Prediger, Luiz Carlos Dias, Annabel C. Murphy, and Peter F. Leadlay*
Abstract: Elaiophylin is an unusual C2-symmetric antibiotic
macrodiolide produced on a bacterial modular polyketide
synthase assembly line. To probe the mechanism and selectivity
of diolide formation, we sought to reconstitute ring formation
in vitro by using a non-natural substrate. Incubation of
recombinant elaiophylin thioesterase/cyclase with a synthetic
pentaketide analogue of the presumed monomeric polyketide
precursor of elaiophylin, specifically its N-acetylcysteamine
thioester, produced a novel 16-membered C2-symmetric mac-
rodiolide. A linear dimeric thioester is an intermediate in ring
formation, which indicates iterative use of the thioesterase
active site in ligation and subsequent cyclization. Furthermore,
the elaiophylin thioesterase acts on a mixture of pentaketide
and tetraketide thioesters to give both the symmetric decaketide
diolide and the novel asymmetric hybrid nonaketide diolide.
Such thioesterases have potential as tools for the in vitro
construction of novel diolides.
dehydratase (DH), and enoylreductase (ER) domains. The
processed intermediates are passed from module to module
until the full-length linear chain is released, most commonly
through the action of a thioesterase/cyclase (TE) domain.[2]
The directness of the link between the PKS gene sequence
and the chemical structure of the end product has revolu-
tionized our view of the evolution of antibiotic biosynthesis,[3]
and has stimulated ongoing efforts to expand polyketide
structural diversity by reprogramming modular assembly
lines.[4] It is particularly important to understand the specific-
ity of chain-terminating TE domains, since these enzymes
have a controlling influence on whether reprogrammed
polyketide products are efficiently released, and on whether
cyclization is favored over hydrolysis.[5] Previous in vitro work
has been carried out on the TE domain that catalyzes
formation of the siderophore enterobactin[6] and on the
assembly-line TE domains for several nonribosomal peptide
synthetases[7] and the results show that such enzymes have
a fairly relaxed specificity and can be deployed as cyclization
catalysts. The X-ray crystal structures have been determined
for chain-terminating TE domains from the PKS assembly
lines for both macrocyclic polyketides[8] and linear poly-
ketides,[9] thus providing a valuable framework for mecha-
nistic investigation. The ability of several individual poly-
ketide TE domains to catalyze the in vitro macrocyclization
of thioester substrates has also been demonstrated.[5a,c,10]
However, we are still far from a detailed understanding of
the factors that influence specificity and selectivity for these
enzymes.
M
odular type I polyketide synthases (PKSs) are giant
multifunctional enzymes, principally from actinomycete bac-
teria, that use a remarkable assembly-line logic for the
biosynthesis of a diverse array of bioactive natural products,[1]
including a number of clinically valuable antibiotics, immu-
nosuppressants, and anticancer compounds. Each module
contains a ketosynthase (KS), which condenses activated acyl
and malonyl units; an acyltransferase (AT), which specifies
the type of extender unit introduced; and an acylcarrier
protein (ACP), which tethers the growing polyketide chain
while it is processed by optional ketoreductase (KR),
An intriguing and relatively rare variation in the mode of
polyketide release from modular PKS assembly lines leads to
C2-symmetric macrocyclic dilactones, or diolides.[11] C2-sym-
metric diolides of diverse ring size have now been charac-
terized from numerous sources including bacteria,[12] fungi,[13]
and marine animals (or their commensal microorganisms).[14]
A better understanding of the molecular basis for such
catalysis might enable a novel mild chemoenzymatic route to
non-natural analogues of such compounds. We report herein
the cloning, expression, and in vitro dimerizing activity of the
chain-terminating TE domain of the modular PKS multi-
enzyme that synthesizes the 16-membered diolide (1b;
Scheme 1) of elaiophylin,[2c,15] a compound with antibacterial,
antiviral, antifungal, and immunomodulatory activities.
Two alternative mechanisms can be advanced for forma-
tion of the symmetrical diolide aglycone 1b on the polyketide
synthase, as illustrated in Scheme 2. In route 1), initial
nucleophilic attack by the distal hydroxy group of the TE-
bound monomer on the ACP-bound thioester affords the
linear dimer attached to the TE active site ready for
cyclization. In route 2), the TE-bound monomer is attacked
[*] Dr. Y. Zhou, Dr. A. C. Murphy, Prof. P. F. Leadlay
Department of Biochemistry, University of Cambridge
80 Tennis Court Road, Cambridge CB2 1GA (UK)
E-mail: pfl10@cam.ac.uk
Prof. P. Prediger,[+] Prof. L. C. Dias
Institute of Chemistry, State University of Campinas
UNICAMP, C.P. 6154, CEP 13084-971, Campinas SP (Brazil)
[+] Current Address: Faculty of Technology
State University of Campinas
UNICAMP, CEP 13484-332, Limeira, SP (Brazil)
[**] We gratefully acknowledge BBSRC (project grant BB/J007250/1 to
P.F.L.), the European Commission (Marie Curie Fellowship to Y.Z.),
and the University of Cambridge (Herchel Smith Research Fellow-
ship to A.C.M.) and Dr. Katherine Stott (Department of Biochem-
istry, University of Cambridge) for help in AUC analysis. L.C.D.
acknowledges the support of Fundażo de Amparo ꢀ Pesquisa do
Estado de S¼o Paulo (FAPESP, Proc. 2012/04616-3 and 2012/02230-
0). P.F.L. is an International Research Awardee of the Alexander von
Humboldt Foundation.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500401.
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Scheme 1. The structures of elaiophylin 1a, elaiolide 1b, and the
analogues of the monomeric polyketide precursor of elaiolide: tetrake-
tide 2 and pentaketide 3.
Figure 1. HPLC–MS analysis of the products of Ela-TE action on model
substrates. A) Compounds 4 and 5, as well as the hydrolysis product
3a, are generated from 3b by Ela-TE. DEBS-TE exclusively catalyzes
hydrolysis to 3a. B) Compound 4, when purified from the reaction
mixture and re-incubated with fresh Ela-TE, is cyclized into 5.
(Scheme 1) to a novel 16-membered decaketide diolide 5
(Figure 1), and we identify an intermediate that sheds light on
the enzymatic mechanism. Although the tetraketide thioester
2b (Scheme 1) is not itself a substrate for homodimerization,
the substrate flexibility of the elaiophylin TE (Ela-TE) is
further shown by the fact that in the presence of both 2b and
3b, a novel asymmetric nonaketide 6 is formed in addition to
the expected decaketide 5 (Figure 2).
Candidate substrates for the diolide cyclase were obtained
through stereoselective synthesis of analogues of the elaio-
phylin monomeric seco acid (Scheme 1). Tetraketide SNAC
thioester 2b was obtained in 12 steps (overall yield 18.3%),
while pentaketide SNAC thioester 3b was obtained in
13 steps (overall yield 8.0%; Scheme 3 and Supporting
Scheme 2. Alternative mechanisms for the formation of symmetrical
diolide 1b. Route 1): initial nucleophilic attack by the distal hydroxyl
group of the TE-bound monomer on the ACP-bound thioester.
Route 2): the TE-bound monomer is attacked by the distal hydroxy
group of a second monomer tethered to the adjacent ACP domain.
by the distal hydroxy group of the ACP-bound monomer to
give the linear dimer attached to the ACP (“retrotransfer”),
and then the linear dimer is transferred to the vacant TE
active site for cyclization. This retrotransfer or iterative
mechanism (route 2)) has previously been demonstrated for
the cyclization steps of nonribosomal peptide synthetases.[7b,c]
By using N-acetylcysteaminyl thioesters (SNAC thioesters) of
tetraketide and pentaketide analogues of the natural octake-
tide monomers, which in vivo are acted upon by the TE while
tethered to an adjacent ACP domain in the multienzyme
assembly line, we show here that the TE can catalyze
homodimerization of the synthetic pentaketide 3b
Figure 2. HPLC–MS analysis of the products of Ela-TE action on an
equimolar mixture of 2b and 3b. The novel asymmetric 16-membered
macrodiolide nonaketide 6 was produced from 2b and 3b and 5 was
generated from 2b alone.
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Information, Section 3. The chain-terminating TE domain
from the previously-characterized elaiophylin PKS[15] was
obtained from E. coli as a soluble protein of the expected
molecular mass (Figure S1 in the Supporting Information). In
contrast to previously studied TE domains from macrocyclic
PKS multienzymes, which retain a dimeric structure,[8] the
elaiophylin TE was found to be largely monomeric in solution
(Figure S2).
techniques (Supporting Information, Section 4.2); as well as
the hydrolysis product 3a. These were accompanied by
a further species eluted after 20.5 min (Figure 1A), the
concentration of which initially rose and then levelled off
during the incubation (Figure S3). The structure of this
species, as determined by HRMS and 1D- and 2D-NMR,
corresponded to the linear dimer 4 (Supporting Information,
Section 4.1). Notably, dimerization of 3b gave only the
symmetrical 16-membered macrodiolide, as found in natural
1b, with no evidence of regioisomers with a different ring size
being formed. We also tested the chain-terminating cyclase/
thioesterase from the erythromycin pathway (DEBS-TE)[16]
as a potential catalyst for the dimerization of 3b, but this
enzyme exclusively catalyzed hydrolysis to 3a
(Figure 1A).
Incubation of 3b (3 mm) with Ela-TE (40 mm) in 0.1m
potassium phosphate buffer (pH 8.2) containing 10%
DMSO produced, in
a time- and enzyme-dependent
manner, the symmetric decaketide diolide 5, the structure of
which was confirmed by HRMS and 1D- and 2D-NMR
When 4 was purified from the reaction
mixture and re-incubated with fresh Ela-TE, it
was cyclized into 5 (Figure 1B), a result con-
sistent with 4 being an essential intermediate in
the macrocyclization of 3b. As a control, we
separately determined that purified 5 was stable
to hydrolysis by Ela-TE under these experimen-
tal conditions (data not shown). These observa-
tions show that the TE is competent to catalyze
both the ligation of two monomeric polyketide
chains and subsequent diolide formation and
they support the mechanism of route 2)
(Scheme 2) for macrodiolide formation in vitro
since route 1) would not generate 4. We propose
that the same iterative mechanism operates
in vivo, especially since the published structures
for dimeric PKS TE domains[8] reveal that
functional communication between the TE
active sites is highly improbable.
In contrast to pentaketide analogue 3b, the
tetraketide 2b yielded only the hydrolysis prod-
uct 2a upon incubation with Ela-TE (data not
Scheme 3. Synthesis of tetraketide 2b and pentaketide 3b as model substrates for Ela-
TE. Reagents and conditions: A) a) i. nBu2BOTf, TEA, CH2Cl2, À788C, ii. EtCHO,
À788C to 08C, 3 h, 65–68% (ds>95:5). b) LiBH4, THF, MeOH, 45 mins, 08C. c) 1-
(dimethoxymethyl)-4-methoxybenzene, CSA, CH2Cl2, RT, 93% (for two steps).
d) DIBAL, CH2Cl2, 45 mins, 08C, 99%. e) TsCl, DMAP, TEA, CH2Cl2, RT, 4 h, 90%.
f) LiBH4, THF, 08C to RT, 24 h, 85%. g) OsO4, NMO, buffer pH 7, THF/acetone, 5 h,
RT. h) NaIO4, buffer pH 7, THF, 12 h, (86% for two steps). i) i. LiHMDS, THF, À788C
to À258C, 30 mins, ii. (E)-ethyl 4-(diethoxyphosphoryl)but-2-enoate, THF, À788C to
À258C, 84%. j) KOH, EtOH/H2O, 12 h, RT, 99%. k) N-(2-mercaptoethyl)acetamide,
DCC, HOBt, DMF, 08C to RT, 12 h, 67%. l) DDQ, buffer pH 7, DCM, 08C, 80%.
B) a) i. nBu2BOTf, DIPEA, CH2Cl2, À108C, ii. À788C, (S)-3-((4-methoxybenzyl)oxy)-2-
methylpropanal, 70%(ds>95:5). b) N,O-dimethylhydroxylamine hydrochloride, Me3Al,
THF, 6 h, 08C to RT, 72%. c) TBSOTf, CH2Cl2, 2,6-lutidine, 1 h, 08C to RT, 79%.
d) EtMgBr, THF, 5 h, 08C, 78%. e) HF·Py, THF, 08C to RT, 12 h, 95%. f) Me4NBH-
(OAc)3, MeCN, AcOH, 12 h, À308C to À208C, 88%. g) TBSOTf, 2,6-lutidine, CH2Cl2,
1 h, 08C to RT, 87%. h) DDQ, buffer pH 7, CH2Cl2, 08C, 82%. i) (COCl)2, DMSO, TEA,
CH2Cl2, À788C, 2 h, 86%. j) i. LiHMDS, THF, À788C to À258C, 30 mins, ii. (E)-ethyl
4-(diethoxyphosphoryl)but-2-enoate, THF, À788C to À258C, 2 h, 94%. k) KOH, EtOH/
H2O, 12 h, RT, 99%. l) N-(2-mercaptoethyl)acetamide, DCC, HOBt, DMF, 08C to RT,
12 h. m) HF·Py, THF, 08C to RT, 12 h, 51% (two steps). ds=diastereoselectivity.
TEA=triethylamine, CSA=10-camphorsulfonic acid, DIBAL=diisobutylaluminum hy-
dride, Ts=4-toluenesulfonyl, DMAP=4-dimethylaminopyridine, NMO=N-methylmor-
pholine-N-oxide, DCC=1,3-dicyclohexylcarbodiimide, HOBt=1-hydroxybenzotriazole,
DMF=N,N-dimethylformamide, DDQ=2,3-dichloro-5,6-dicyano-1,4-benzoquinone,
DIPEA=N,N-diisopropylethylamine, TBSOTf=tert-butyldimethylsilyl trifluoromethane-
sulfonate, LiHMDS=lithium hexamethyldisilazane.
shown). Interestingly, when an equimolar mix-
ture of 2b and 3b was incubated with Ela-TE
under the same conditions, the asymmetric 16-
membered macrodiolide nonaketide 6, the
structure of which was confirmed by HRMS
and 1D- and 2D-NMR (Supporting Information,
Section 4.3), was produced in addition to 5 and
in comparable amounts (Figure 2).
To the best of our knowledge, this is the first
example of a “hybrid” macrodiolide polyketide
produced enzymatically in vitro. In this experi-
ment, LC–MS revealed the presence of a species
with the mass predicted for a linear nonaketide
thioester with the same retention time as 4
(Figure S4). However insufficient material was
available to allow NMR analysis. Further work
will thus be required to determine the exact
course of the reaction.
Previous structural studies on PKS TE
domains catalyzing either hydrolysis[9] or macro-
cyclization[8] have identified the active site as
lying within an unusual channel that traverses
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Received: January 15, 2015
Revised: February 4, 2015
Published online: && &&, &&&&
Keywords: biosynthesis · diolides · elaiophylin ·
.
polyketide synthase · thioesterase
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Communications
Biosynthesis
Y. Zhou, P. Prediger, L. C. Dias,
A. C. Murphy,
P. F. Leadlay*
&&&& — &&&&
Macrodiolide Formation by the
Thioesterase of a Modular Polyketide
Synthase
Think again: The thioesterase/cyclase
enzyme of the elaiophylin polyketide
synthase catalyzes symmetric diolide
formation in vitro from a synthetic pen-
taketide substrate by an iterative mecha-
nism. Unexpectedly, a tetraketide that is
not itself a substrate can be co-opted in
the presence of the pentaketide to pro-
duce an asymmetric macrodiolide.
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
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