Polyketide synthase thioesterases catalyze rapid hydrolysis of peptidyl thioesters

Article abstract of DOI:10.1016/j.bmcl.2009.01.040

Polyketide synthase (PKS) thioesterases (TEs) catalyze the macrocyclization of linear acyl chains into macrolactones. Herein we show that peptide based substrates are processed by PKS TEs with greater catalytic efficiency than more native like acyl substrates. This result strengths the link between PKS and non-ribosomal peptide synthetase systems and provides a new tool for studying PKS TEs.

Full text of DOI:10.1016/j.bmcl.2009.01.040

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1413–1415
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Polyketide synthase thioesterases catalyze rapid hydrolysis
of peptidyl thioesters
Meng Wang a, Peter Opare a, Christopher N. Boddy a,b,
*
a Department of Chemistry, Syracuse University, Syracuse, NY 13244-4100, USA
b Department of Chemistry, University of Ottawa, Ottawa, Ont., Canada K1N 6N5
a r t i c l e i n f o
a b s t r a c t
Article history:
Polyketide synthase (PKS) thioesterases (TEs) catalyze the macrocyclization of linear acyl chains into
macrolactones. Herein we show that peptide based substrates are processed by PKS TEs with greater cat-
alytic efficiency than more native like acyl substrates. This result strengths the link between PKS and non-
ribosomal peptide synthetase systems and provides a new tool for studying PKS TEs.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 November 2008
Revised 9 January 2009
Accepted 13 January 2009
Available online 19 January 2009
Keywords:
Polyketide
Peptidyl thioester
Thioesterase
Enzyme kinetics
Substrate specificity
The thiotemplate-directed biosynthesis strategy is shared by
modular polyketide synthases (PKSs) and non-ribosomal peptide
synthetases (NRPSs).1,2 Numerous important pharmaceutical
crystallographic analysis shows that both classes of TE belong to
/b hydrolase family.21–24 In vitro kinetic characterization of TEs
has shown them to be substrate specific with specificity constants
varying over orders of magnitude. Recent studies show that sub-
strate selectivity in both NRPS and PKS TEs is mediated through
a
3–5
agents are produced by PKSs or NRPSs, such as erythromycin (1),
pimaricin (2), 6,7 epothilone (3), 8–10 tyrocidine,11,12 and gramicidin
S.13 Both classes of enzymes have a modular architecture and func-
tion like an assembly-line, incorporating simple building blocks
such as malonyl-CoA (PKS) or amino acid (NRPS) into a growing
linear chain.
The final step in both PKS and NRPS biosynthesis is often mac-
rocyclization, which is catalyzed by thioesterases (TE) present at
the C-terminus of PKSs and NRPSs. Macrocyclization is crucial for
the bioactivity and the pharmaceutical utility of the compounds
produced (Fig. 1).14 These striking similarities in the biosynthetic
strategies for the formation of polyketides and non-ribosomal pep-
tides have led us to study the homology between PKS and NRPS
TEs.
PKS and NRPS TEs are mechanistically homologous, catalyzing
macrocyclization via a two-step mechanism.15 The first step, which
is rate determining in vitro,16–18 is the acylation of an active site
serine by a linear thioester substrate. This is followed by either
intramolecular nucleophilic attack leading to macrocyclization14
or hydrolysis leading to the linear carboxylate.19,20
PKS and NRPS TEs are structurally and functionally homologous.
Both show high primary sequence homology and high-resolution
* Corresponding author. Tel.: +1 613 562 5800/8970; fax: +1 613 562 5170.
E-mail address: cboddy@uottawa.ca (C.N. Boddy).
Figure 1. Structures of erythromycin, pimaricin, epothilone D, and virginiamycin
M. The bonds generated by the thioesterase domains are highlighted.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.040

1414
M. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1413–1415
hydrophobic interactions between the substrates and the enzyme
binding pocket.25–28
peptide substrates, including amide bonds, epimerized proteino-
genic amino acids, and non-proteinogenic amino acids. All com-
pounds were synthesized by standard solution phase organic
synthesis techniques and were purified to homogeneity as mea-
sured by 1H NMR spectroscopy.
The excised recombinant thioesterase domains from the pimar-
icin, epothilone, and 6-deoxyerythronolide B pathways, Pim TE,
Epo TE, and DEBS TE, respectively, were over-expressed in Esche-
richia coli and isolated in high purity by affinity chromatography.
Steady state kinetic parameters were determined for the thioester-
ase catalyzed hydrolysis of substrates 6, 8, 10, 12, 14, 16, 19, and
20 by quantifying the production of free thiol using Ellman’s
reagent.16,34
Many natural products are produced by hybrid PKS/NRPS sys-
tem.19,29,30 TE substrates produced by these hybrid systems pos-
sess functional groups typical of both polyketide and peptide. For
example, the DKxanthene,19 and virginiamycin M31 (4) biosyn-
thetic pathways, which are predominantly comprised of PKS mod-
ules, also contain multiple NRPS modules. To cyclize these natural
products, the respective TEs need to accommodate functional
groups from both polyketides and peptides.
While highly homologous, some data has been emerging sug-
gesting differences between PKS and NRPS TEs. In general it ap-
pears that NRPS TEs are substantially more kinetically competent
than PKS TEs. Specificity constant (kcat/KM) and turnover rate (kcat
)
Due to the poor solubility of the substrates in the reaction buf-
fer and the high KM,16,34 it was not possible to determine kcat and
the KM independently for all substrates. For 16, 19, and 21 with
the Epo TE and 16 with the Pim TE kcat and KM could be determined
independently. The specificity constant (kcat/KM) was determined
for all remaining substrates. In these cases KM is estimated at great-
er than 2 mM as enzyme saturation was not reached with 4 mM
substrate concentrations.
Specificity constants for TE-mediated hydrolysis of NRPS-like
peptidyl substrates by PKS TEs are given in Table 1. The Epo TE
was able to hydrolyze 16 with kcat = 1.82 0.06 sÀ1 and KM = 0.40
0.06 mM, 19 with kcat = 3.1 0.4 sÀ1 and KM = 1.3 0.5 mM, and 21
with kcat = 19.2 2.2 sÀ1 and KM = 2.9 0.6 mM. The Pim TE hydro-
lyzed 16 with kcat = 1.2 0.2 sÀ1 and KM = 0.8 0.1 mM.
Our kinetic data shows that amide containing peptidyl sub-
strates are processed by PKS TEs. The specificity constants ob-
served for the peptidyl substrates are greater than or equal to
specificity constants determined for more native-like polyacetate
and polypropionate substrates.34 The KMs observed and estimated
for these peptidyl substrates were comparable to the KMs for poly-
ketide-like substrates.33,35 This suggests that an increase in kcat is
responsible for the greater kinetic efficiency in the processing of
peptidyl substrates versus polyketide-like substrates by PKS TEs.
for in vitro substrate processing are generally much greater for
NRPS TEs. Additionally the Michaelis constants (KM) for NRPS TEs
tend to be much lower than those seen for PKS TEs.
To further probe these two key macrocyclizing enzymes, we set
out to investigate the substrate tolerance of the PKS TEs with
NRPS-like peptidyl substrates. The TE-mediated hydrolysis of sim-
ple amino acid and dipeptide thioesters was kinetically character-
ized for the TEs from the pimaricin (Pim TE),32 epothilone (Epo
TE),33 and 6-deoxyerythronolide B (DEBS TE)16 biosynthetic path-
ways. We show that PKS TEs can efficiently process these amide-
containing peptide-like substrates and that these substrates are
processed with much higher specificity constants than any other
PKS TE substrates characterized to date. This observation further
strengthens the link between PKS and NRPS systems.
A panel of eight amide containing, N-acetyl cysteamine (SNAC)
thioester substrates was synthesized to probe the substrate speci-
ficity of PKS TEs. For these substrates, no intramolecular nucleo-
philes are available to undergo TE catalyzed cyclization.
Therefore, after loading the substrates onto the thioesterase to
form the acyl–enzyme intermediate, only TE catalyzed hydrolysis
can occur. The substrate panel can be divided into two groups. Sub-
strates 6, 8, 10, 12, 14, and 16 are thioesters activated N-tert-butyl
carbamate protected amino acids. Substrates 6, 8, 10, and 12 vary
Mono-substitution at the a-position appears to be tolerated by
in the substitution and stereochemistry at the
a
-position. Sub-
the DEBS and Epo TE but not the Pim TE. The S and R enantiomers
of alanine, 10 and 8, respectively, are efficiently hydrolyzed by
DEBS and Epo TE. Neither of these substrates is hydrolyzed by
the Pim TE. This is surprising since a previous study had shown
strates 14 and 16 differ in the location of the amide linkage. Sub-
strates 19 and 21 are dipeptides and have different
stereochemistry at d-position (Fig. 2). The chemical diversity of this
panel represents multiple structural elements from non-ribosomal
that Pim TE can hydrolyze
a-methylated polypropionate sub-
O
O
O
O
R
H
N
H
N
H
N
H
N
Boc
R
Boc
R
Boc
R
Boc
5 R = OH
6 R = SNAC
7 R = OH
8 R = SNAC
9 R = OH
10 R = SNAC
11 R = OH
12 R = SNAC
EDC
HSNAC
(61 %)
EDC
HSNAC
(79 %)
EDC
HSNAC
(71 %)
EDC
HSNAC
(88 %)
O
O
O
Boc
N
Ph
N
H
R
R
H
13 R = OH
14 R = SNAC
15 R = OH
16 R = SNAC
EDC
EDC
HSNAC
(94%)
HSNAC
(77)%)
O
O
O
O
(D)-BocAla-OH
1. LiOH
H
H
HOBt, EDC, Et3N
Boc
Boc
N
2. EDC, HSNAC
(10%)
H2N
Boc
N
N
H
OMe
OMe
OMe
N
H
SNAC
(55%)
O
O
O
17
18
19
(L)-BocAla-OH
HOBt, EDC, Et3N
O
1. LiOH
2. EDC, HSNAC
H
N
H
N
Boc
(34%)
N
H
N
SNAC
H
(10%)
O
20
21
Figure 2. Synthesis of substrates used to investigate the substrate specificity of PKS thioesterase domains.

M. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1413–1415
1415
Table 1
facile, the substrate tolerance and specificity of PKS TEs can be
probed much more rapidly. Incorporation of amide linkages into
complex TE substrates will also facilitate study of TE-mediated
macrocyclization by allowing easy synthetic access to libraries of
substrates capable of undergoing macrocyclization. Lastly this re-
sult further strengthens the linkage between PKS and NRPS biosyn-
thetic systems.
Steady state kinetic perimeters for thioesterase catalyzed hydrolysis.
DEBS TE kcat/KM
(MÀ1 SÀ1
Epo TE kcat/KM
(MÀ1 SÀ1
Pim TE kcat/KM
(MÀ1 SÀ1
)
)
)
6
8
7.0 0.4
0.50 0.01
0.88 0.03
NR
7.75 0.19
1.47 0.07
0.80 0.11
0.39 0.03
145 10
12.5 0.5
91.3 2.9
1.29 0.05
NR
NR
10
12
14
16
19
21
NR
NR
24.2 1.2
75.1 11.5
38.8 14.6
110.0 27.2
1.14 0.06
23.3 3.6
NR
Acknowledgments
NR
This research was supported by Syracuse University and the
University of Ottawa.
NR, not reactive.
Supplementary data
strates.32 We proposed that peptidyl substrates adopt a different
binding mode preventing substrates with -substitution from
loading onto the active site serine. The Epo TE shows a 10-fold
preference for the S configuration (10 versus 8); however, no sub-
stituents are found at the C2 position in the epothilone family of
products. The gem dimethyl compound 12 is not processed by
a
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.040.
References and notes
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