Enediyne polyketide synthases stereoselectively reduce the β-ketoacyl intermediates to β- D -hydroxyacyl intermediates in enediyne core biosynthesis

Article abstract of DOI:10.1021/ol501767v

PKSE biosynthesizes an enediyne core precursor from decarboxylative condensation of eight malonyl-CoAs. The KR domain of PKSE is responsible for iterative β-ketoreduction in each round of polyketide chain elongation. KRs from selected PKSEs were investiga

Full text of DOI:10.1021/ol501767v

Letter
pubs.acs.org/OrgLett
Enediyne Polyketide Synthases Stereoselectively Reduce the
β‑Ketoacyl Intermediates to β‑D‑Hydroxyacyl Intermediates in
Enediyne Core Biosynthesis
Hui-Ming Ge,† Tingting Huang,† Jeffrey D. Rudolf,† Jeremy R. Lohman,† Sheng-Xiong Huang,†
Xun Guo,† and Ben Shen*,†,‡,§
†Department of Chemistry, ‡Department of Molecular Therapeutics, and §Natural Products Library Initiatives, The Scripps Research
Institute, Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: PKSE biosynthesizes an enediyne core pre-
cursor from decarboxylative condensation of eight malonyl-
CoAs. The KR domain of PKSE is responsible for iterative β-
ketoreduction in each round of polyketide chain elongation.
KRs from selected PKSEs were investigated in vitro with β-
ketoacyl-SNACs as substrate mimics. Each of the KRs reduced
the β-ketoacyl-SNACs stereoselectively, all affording the
corresponding β-D-hydroxyacyl-SNACs, and the catalytic
efficiencies (kcat/KM) of the KRs increased significantly as the chain length of the β-ketoacyl-SNAC substrate increases.
olyketides are a structurally diverse family of natural
products with a broad range of biological activities. While
P
their biosynthesis is mechanistically similar to that of fatty acids,
reductive modifications of the β-keto groups to different extents
at each cycle of polyketide chain elongation differ from that for
fatty acid biosynthesis and result in polyketides bearing a
variety of functionalities at the β-positions. Among polyketides,
the enediyne natural products are some of the most potent
antitumor antibiotics known to date.1 Common to the
enediyne natural products is a 9- or 10-membered carbocyclic
enediyne core. Biosynthesis of the carbon skeleton of enediyne
cores from eight molecules of malonyl-CoA is catalyzed by the
enediyne polyketide synthase (PKSE).2,3 PKSEs are iterative
type I PKSs that are characterized with six domains, ketoacyl
synthase (KS), acetyltransferase (AT), acyl carrier protein
(ACP), ketoreductase (KR), dehydratase (DH), and phospho-
pantetheinyl transferase (PPT) (Figure 1A).2,3 While this
domain organization is quite distinct from other type I PKSs, it
is absolutely conserved among all PKSEs known to date (Figure
S1, Supporting Information (SI)).
Figure 1. Enediyne polyketide synthase (PKSE) and its proposed roles
in 9- and 10-membered enediyne biosynthesis. (A) Domain
organization of PKSEs: KS, ketoacyl synthase; AT, acetyltransferase;
ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase; and
PPT, phosphopantetheinyl transferase. (B) A unified model for
enediyne biosynthesis featuring common PKSE chemistry with
accessory enzyme-directed pathway divergence to 9- and 10-
membered enediyne cores. The KR domain and its catalyzed
chemistry are highlighted in red. MCoA, malonyl-CoA; TE,
thioesterase. See Figure S1 (SI) for structures of C-1027, calicheamicin
(CAL), dynemicin (DYN), kedarcidin (KED), maduropeptin (MDP),
neocarzinostatin (NCS), and uncialamycin (UCM).
While the indispensable role PKSE plays in enediyne natural
product biosynthesis has been unambiguously established,2 it
remains elusive how the enediyne cores are constructed and
what determines the biosynthetic divergence between 9- versus
10-membered enediynes.3 Based on phylogenetic analysis of
the known PKSEs, we had previously proposed that the PKSEs
for 9- and 10-membered enediyne cores were distinct, and this
distinction might be critical to the alternate folding and early
modification steps that lead to the divergence of the 9- and 10-
membered enediynes.3a,b This proposal was supported by in
vivo studies, in which Streptomyces globisporus SB1005, a ΔsgcE
mutant strain of the C-1027 producer S. globisporus, and
Streptomyces carzinostaticus SB5002, a ΔncsE mutant strain of
the neocarzinostatin (NCS) producer S. carzinostaticus, can be
functionally complemented to restore C-1027 or NCS
production, respectively, by PKSE genes from selected 9-
Received: June 18, 2014
Published: July 14, 2014
© 2014 American Chemical Society
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Letter
membered, but not 10-membered, enediyne pathways.3c
Subsequent in vitro studies of selected PKSEs, alone or in
combination with the associated thioesterases (TEs), revealed
that PKSEs most likely biosynthesized a common intermediate
that was then morphed into the 9- and 10-membered scaffolds
by pathway-specific accessory enzymes, and the reconstituted
PKSE-TE of both 9- and 10-membered enediyne core origin
afforded enzymatic synthesis of the same set of products, most
notably a heptaene (Figure 1).4 These in vitro findings were
further supported by (i) heptaene production upon expressing
selected pksE genes of both 9- and 10-membered enediyne core
origin with their cognate and noncognate TE genes in both E.
coli and Streptomyces and (ii) detection of heptaene from
selected native producers of both nine- and ten-membered
enediyne natural products (Figure 1).3c,d
Despite these advances, it is far from certain that the
stereochemistry of the double bonds deduced from the isolated
heptaene could be directly correlated to the PKSE-tethered
precursor that is ultimately morphed into the 9- or 10-
membered enediyne cores. The KR domain catalyzes the
iterative β-ketoreduction in each round of polyketide chain
elongation, affording the PKSE-tethered β-hydroxyacyl inter-
mediates that are subsequently dehydrated by the DH domain
to the double bonds (Figure 1B). For bacterial noniterative
type I PKSs, the stereochemistry of β-hydroxyacyl intermedi-
ates destines the DH domain in the ensuing dehydration step
to afford a cis- or trans-double bond in the growing polyketide
products.5,6 Thus, it is tempting to speculate that the
stereochemistry of PKSE-tethered β-hydroxyacyl intermediates
could dedicate the geometry of the resultant double bonds, the
difference of which in the PKSE-tethered full length polyene
products could impact on eventual formation of the 9- or 10-
membered enediyne core by the pathway-specific accessory
enzymes.
Herein, we report in vitro characterization of KRs from seven
selected PKSEs using β-ketoacyl-N-acetylcysteamines (SNACs)
with varying acyl chain lengths as substrate mimics.
Remarkably, each of the KRs reduced the β-ketoacyl-SNACs
stereoselectively, all affording the corresponding β-D-hydrox-
yacyl-SNACs, and the catalytic efficiencies (kcat/KM) of the KRs
increased significantly as the chain length of the β-ketoacyl-
SNAC substrate increases.
The KR domains from four nine-membered [C-1027,
kedarcidin (KED), maduropeptin (MDP), and neocarzinostatin
(NCS)] and three 10-membered [calicheamicin (CAL),
dynemicin (DYN), and uncialamycin (UCM)] PKSEs were
selected in this study (Figure S1, SI).2 While little is known
about the KR domain of PKSE, KRs of type I PKSs have been
extensively characterized mechanistically and structurally.5−7
These KR domains, averaging ∼466 amino acids, catalyze
stereoselective β-keto reduction of the PKS-tethered β-ketoacyl
intermediates (via the ACP domain), utilizing NADPH. The
KR domains have been further classified into A- or B-type. A-
type KRs, featuring a conserved Trp, generate a β-L-hydroxyacyl
intermediate, and B-type KRs, featuring an LDD motif, afford a
β-D-hydroxyacyl intermediate (Figure 2A); the presence of the
LDD motif in the B-type KRs and the absence of the LDD
motif, accompanied by the presence of the conserved Trp, in
the A-type KRs have often been used to discriminate the two
types of KRs.6,7 The KR domains from PKSEs show high
sequence homology among each other, as well as to known
KRs of type I PKSs, possessing the critical catalytic residues
(Lys, Ser, and Tyr) and the conserved Gly-rich motif for
Figure 2. In vitro assay of KRs of PKSEs for both 9- and 10-membered
enediyne core biosynthesis using β-ketoacyl-SNACs as substrate
mimics revealing the same intrinsic stereoselectivity to afford the β-D-
hydroxyacyl-SNAC products. (A) A- and B-type KRs of type I PKS
affording β-L- or β-D-hydroxyacyl intermediates in polyketide biosyn-
thesis featuring the conserved residue and diagnostic motif (i.e., W for
A-type and LDD for B-type).6,7 ACP, acyl carrier protein. (B) The
synthesized β-ketoacyl-SNACs as substrate mimics (1−3) and β-D-
hydroxyacyl-SNACs (4−6) and β-L-hydroxyacyl-SNACs (7−9) as
authentic standards of the reduced products. (C) In vitro assay of KRs
of PKSEs using β-ketoacyl-SNACs as substrate mimics in the presence
of NADPH. Chiral HPLC analysis of KR-catalyzed stereoselective
reduction of the β-ketoacyl-SNAC substrates 1 (D), 2 (E), and 3 (F)
to the corresponding β-D-hydroxyacyl-SNAC products 4, 5, and 6. (i)
⧫
Authentic standards of the β-D-hydroxyacyl-SNAC ( ) and β-L-
●
hydroxyacyl-SNAC ( ) products; (ii) KR of SgcE; (iii) KR of KedE;
(iv) KR of MdpE; (v) KR of NcsE; (vi) KR of CalE8; (vii) KR of
DynE8; (viii) KR of UcmE.
NADPH binding. However, they lack both the LDD motif and
the conserved Trp residue, the absence of both of which makes
it difficult to classify the KRs of PKSEs into A- or B-type KRs,
thereby predicting the stereochemistry of the resultant β-
hydroxyacyl products (Figure S2, SI).6,7
The boundaries of the KRs of PKSEs were defined by
following those established for the KRs of type I PKSs.5−7 The
seven KRs, i.e., KRs from SgcE (residues 1018−1452), KedE
(residues 1008−1448), MdpE (residues 1033−1466), NcsE
(residues 1055−1488), CalE8 (residues 1025−1448), DynE8
(residues 1011−1437), and UcmE (residues 1003−1444), were
overproduced in E. coli BL21(DE3) (Figures S1, S2, and Table
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S1, SI) as N-terminal His6-tagged fusion proteins. Each of the
KRs was purified to homogeneity and showed as a single band
upon SDS-PAGE analysis consistent with the predicted
molecular weight of each protein (Figure S3, SI).
SI). The calculated kcat/KM for each of the KRs was
summarized in Table 1. While each of the KRs showed similar
Table 1. Pseudo-First-Order Kinetic Parameters of KRs of
Three β-ketoacyl-SNACs with varying chain lengths (i.e., 1,
2, and 3) were next synthesized as substrates to directly test the
activity of KRs in vitro (Figure 2B and Figures S4−S9, SI). β-
Ketoacyl-SNACs are known mimics of the ACP-tethered
growing polyketide intermediates.5−7 The three substrates
were designed to mimic the nascent polyketide intermediates in
the early rounds of chain elongation in enediyne core
biosynthesis, and 1, 2, and 3 mimic the diketide, triketide,
and tetraketide intermediate, respectively (Figure 1B). Since
the stereoselectivity of the selected KRs of PKSEs cannot be
predicted a priori, we also prepared both enantiomers of the
corresponding reduced products, i.e., β-L-hydroxyacyl-SNACs
(4−6) and β-D-hydroxyacyl-SNACs (7−9) (Figure 2B and
Figures S10−S15, SI), as authentic standards for chiral HPLC
analysis, and the absolute stereochemistry of β-hydroxyacyl-
SNACs was independently established by Mosher’s method
(Figures S16−S21, SI).
The enzymatic activity for each of the seven KRs was
investigated by in vitro assay of the three substrate mimics in
the presence of NADPH (Figure 2C). The enzymatic reaction
courses were followed by HPLC analysis for the disappearance
of the substrate (1, 2, or 3) and appearance of the products (4,
5, or 6) and by continuous photometric measurement at 340
nm for the consumption of NADPH (SI). Thus, upon addition
of the KR proteins to reaction mixtures containing the substrate
mimics and NADPH, the absorbance at 340 nm began to
decline immediately, indicative of NADPH consumption
(Figure S22, SI), and HPLC analysis of the assay mixtures
confirmed time-dependent reduction of the substrates to
specific products. Close examination of the reduction products
by chiral HPLC revealed that the seven KRs all reduced the β-
keto group in 1, 2, or 3 efficiently to the corresponding β-D-
hydroxyacyl-SNACs 4, 5, or 6, respectively, the identities of
which were unambiguously established by LCMS and chiral
HPLC analysis in comparison with synthetic standards (Figure
2D−F). These findings provide direct experimental evidence,
supporting that the KRs in PKSEs can iteratively reduce the β-
ketoacyl intermediates in each round of chain elongation, and
revealing for the first time that, regardless of the origin of the
PKSEs, the KRs exhibit the same intrinsic stereoselectivity
toward the growing β-ketoacyl intermediates, affording the β-D-
hydroxyacyl intermediates in the biosynthesis of both 9- and
10-membered enediyne cores.
the Selected PKSEs toward the Three β-Ketoacyl-SNACs
a
kcat/KM (s−1 M−1
)
(β-ketoacyl-SNACs)
KR (PKSE)
1
2
3
SgcE
12
2
17
27
15
22
28
13
18
5
3
2
2
2
1
1
240 34
110 12
KedE
MdpE
NcsE
6.1 2.0
5.7 0.8
8.4 1.5
140
8
170 20
210 24
160 34
133 31
CalE8
DynE8
UcmE
15
2
4.7 0.6
3.1 0.7
a
Based on the linear fit of the limited case of the Michaelis−Menten
model (v = (kcat/KM)[S][E]0) for low substrate concentration (where
[S] ≪ KM).5d Data are reported as means of triplicate experiments
(Figure S23, SI).
catalytic efficiencies (kcat/KM) toward the same substrate, the
catalytic efficiencies of the KRs all increased significantly as the
chain length of the β-ketoacyl-SNAC substrates increases. Such
properties in fact would be consistent with the processive
mechanism of polyketide biosynthesis so that all elongating
intermediates could rapidly proceed to completion without
stalling the biosynthetic machinery. It would be very interesting
to investigate if the other domains (KS and DH) of PKSE have
similar intrinsic catalytic properties.
Classification of KRs into A- or B-type according to the
conserved residues and motifs (Figure 2A) was based mainly on
bioinformatics analysis of noniterative type I PKSs of bacterial
origin.6,7 Each KR acts once, during each cycle of chain
elongation, to furnish a β-D- or β-L-hydroxy group in the
growing polyketide intermediate, and this model has been
supported by site-directed mutagenesis studies and X-ray
structural analysis of several KRs. The conserved Trp in the
A-type KRs and the LDD motif in the B-type KRs reside on
opposite sides of the active site groove and control the entrance
of substrates into the active site from different sides. The
guided entrance of substrates positions the β-keto group in
opposite orientations relative to the pro-4S hydride of NADPH
and, as such, stereoselective attack of the β-keto group by the
hydride results in a β-D- or β-L-hydroxyacyl product.6,7c,d PKSEs
are iterative type I PKSs that are of bacterial origin,3 and
iterative type I PKSs for aromatic polyketide biosynthesis in
bacteria are also known.8 Although the KRs of iterative PKSs
show significant sequence homology to KRs of their non-
iterative counterparts, the diagnostic Trp residue and LDD
motif for A- or B-type classification are not conserved (Figure
S2, SI). The stereochemistry of KRs of iterative type I PKSs
therefore cannot be similarly predicted according to the model
of KRs of noniterative type I PKSs, as exemplified by the
current study of the KRs of PKSEs. Similar findings have also
been observed for fungal iterative type I PKSs.9 It would
therefore surely be rewarding to compare and contrast the KR
structures of iterative and noniterative type I PKSs, thereby
deciphering the molecular control of the β-hydroxy stereo-
chemistry in polyketide biosynthesis.
The three β-ketoacyl-SNACs with varying chain lengths also
provided an excellent opportunity to probe the relative rates for
the reductive modification steps at each round of chain
elongation, and such rates could have fundamental impact on
the net metabolic pathway flux according to the processive
mechanism for polyketide biosynthesis. Although poor
substrate solubility prevented us from using substrate
concentrations well above the expected KM values to determine
the single-substrate kinetic constants, the apparent kcat/KM
values for each of the KRs were obtained from the linear fit
of the limiting case of the Michaelis−Menten model (v = (kcat/
KM)[S][E]0) for low substrate concentration (where [S] ≪
KM).5d Thus, each of the KRs was assayed with the three
substrate mimics, and the enzymatic reactions were followed
spectrophotometrically by continuously following the absorb-
ance at 340 nm to measure NADPH consumption (Figure S23,
KR of iterative type I PKS must act iteratively to catalyze
multiple rounds of β-ketoreduction in polyketide biosyn-
thesis.3,8,9 Although one would expect each ketoreduction to
occur with the same stereochemistry, as supported by the
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(b) Zazopoulos, E.; Huang, K.; Staffa, A.; Liu, W.; Bachmann, B. O.;
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current study of the KRs of PKSEs for the first three rounds of
chain elongation, caution has to be taken not to overinterpret
this finding, and substrate-dependent stereoselectivity has
indeed been reported for a KR of fungal iterative PKS.9 For
type I PKSs, it is the stereochemistry of β-hydroxyacyl
intermediates, rather than the cognate DH domain, that has
been correlated to the double bond geometry of the resultant
elongating polyketide products5,6,7c,d If this would be the same
for PKSEs, the β-D-hydroxyacyl products from all the KRs
examined in the current study would suggest polyene
intermediates with all double bonds in trans configuration in
the biosynthesis of both nine- or ten-membered enediyne core,
a provocative prediction that begs for experimental verification.
Finally, the current study unambiguously established that the
KRs of PKSEs of both 9- and 10-membered enediynes generate
identical β-D-hydroxyacyl intermediates at least in the first three
rounds of chain elongation (Figures 1B and 2C−F). These
findings further support the previous conclusion that PKSE
chemistry does not direct biosynthetic divergence between 9-
and 10-membered enediynes.3,4
(6) Keatinge-Clay, A. T. Nat. Prod. Rep. 2012, 29, 1050−1073.
(7) (a) Reid, R.; Piagentini, M.; Rodriguez, E.; Ashley, G.;
Viswanathan, N.; Carney, J.; Santi, D. V.; Hutchinson, C. R.;
McDaniel, R. Biochemistry 2003, 42, 72−79. (b) Caffrey, P.
ChemBioChem 2003, 4, 654−657. (c) Keatinge-Clay, A. Chem. Biol.
2007, 14, 898−908. (d) Zheng, J. T.; Taylor, C. A.; Piasecki, S. K.;
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ASSOCIATED CONTENT
* Supporting Information
Complete description of materials and methods, Table S1,
Figures S1−S23. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
(8) Zhang, Q.; Pang, B.; Ding, W.; Liu, W. ACS Catal. 2013, 3,
1439−1447.
AUTHOR INFORMATION
Corresponding Author
■
(9) Zhou, H.; Gao, Z. Z.; Qiao, K. J.; Wang, J. J.; Vederas, J. C.; Tang,
Y. Nat. Chem. Biol. 2012, 8, 331−333.
*E-mail: shenb@scripps.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Y. Li, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences, Beijing, China, for the
wild-type Streptomyces globisporus strain that produces C-1027
and Dr. Julian Davies, University of British Columbia,
Vancouver, Canada, for the wild-type Streptomyces uncialis
DCA2648 strain that produces uncialamycin. This work was
supported in part by NIH Grant No. CA78747.
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