Production of octaketide polyenes by the calicheamicin polyketide synthase CalE8: Implications for the biosynthesis of enediyne core structures

Article abstract of DOI:10.1021/ja904391r

(Chemical Equation Presented) Enediyne antibiotics are categorized according to the presence of either a 9- or 10-membered ring within their polyketide-derived core structures. Recent literature reports have favored the notion that biosynthetic divergence

Full text of DOI:10.1021/ja904391r

Published on Web 08/18/2009
Production of Octaketide Polyenes by the Calicheamicin Polyketide Synthase
CalE8: Implications for the Biosynthesis of Enediyne Core Structures
Katherine Belecki, Jason M. Crawford,† and Craig A. Townsend*
Department of Chemistry, The Johns Hopkins UniVersity, Baltimore, Maryland 21218
Received May 30, 2009; E-mail: ctownsend@jhu.edu
Enediyne antitumor antibiotics are notable for their distinctive
molecular architecture and unique DNA cleavage mechanism.1,2
Formation of an aryl diradical capable of abstracting two hydrogen
atoms, one from each strand of DNA, ultimately results in double-
stranded DNA scission. While total syntheses of several of these
antibiotics have been achieved,3-5 knowledge of their origins in
nature had, until recently, been limited to classical labeling studies
revealing their polyketide folding patterns.6-8 Five biosynthetic
gene clusters are now known for this class of natural products,9-13
but the key steps of enediyne core assembly remain undefined.
A family of highly reducing iterative type I polyketide synthase
(PKS) enzymes is responsible for synthesizing the carbon skeletons
of the enediyne core structures.9,10 These PKSs are highly conserved
among the enediynes yet are quite distinct from other PKSs,
especially in their domain organization (Figure 1A).14 The enediyne
natural products are classified as either 9- or 10-membered,
according to the size of the smallest carbocyclic ring containing
the triple bonds. The neocarzinostatin (NCS) chromophore and
I
calicheamicin γ1 are prominent examples from each group (Figure
1B). Determining the point at which the biosynthetic pathways to
these two families diverge is an important first step in understanding
this biosynthetic problem.
Recent reports15,16 of two different metabolites isolated from
Figure 1. (A) Enediyne PKS domain organization. AT, acyl transferase;
heterologous coexpression experiments of enediyne PKSs (one from
CP, acyl carrier protein; PPT, phosphopantetheinyl transferase. KS, KR,
each of the two structural categories) with their separately encoded
thioesterases have led to the suggestion that the 9- and 10-membered
enediynes diverge biosynthetically at the PKS stage (Figure 1C).
We now report the observation of both of these products arising
from a single biosynthetic system, calling into question both the
assertion of divergence at this point and the legitimacy of either of
these products as a true precursor on the path to calicheamicin.
Heptaene 1 was isolated by Shen and co-workers15 from the
heterologous coexpression of NcsE, the NCS enediyne PKS, and
the putative thioesterase NcsE10, marking the first reported product
of an enediyne PKS. This hydrocarbon polyene was also found
when homologues SgcE and SgcE10, located in the biosynthetic
gene cluster for the 9-membered enediyne C-1027, were coex-
pressed. As a consequence of these observations, 1 was purported
to be a common intermediate in the biosynthetic pathways to the
9-membered enediynes.
and DH are defined in the text. (B) Selected enediyne anticancer antibiotics.
(C) Current literature proposals for enediyne biosynthesis. Polyenes 1 and
2, isolated from coexpression experiments, have been advocated as
precursors to their respective enediyne families. The olefin stereochemistry
depicted has not been verified for all double bonds.
the purified SgcE and CalE8 proteins themselves as further proof
of differentiation occurring at this point.
We have cloned the full length enediyne PKS calE8 and its
associated thioesterase calE7 from the genomic DNA of Mi-
cromonospora echinospora ssp. calichensis for in Vitro reconstitu-
tion experiments with purified enzymes. As has been noted by
others, CalE7 assists in the efficient release of enzyme-bound
polyketides.16,17 Purification of CalE8 following heterologous
expression in Escherichia coli yields a bright yellow solution of
soluble, active protein. We found the UV/vis spectrum to reveal
both a broad absorbance in the 350-500 nm range and the fine
structure indicative of a hydrocarbon polyene system, similar to
that seen with SgcE (Figure 2A). This feature is harder to discern
at lower concentrations but is present nonetheless.
Cysteine 211, identified as a catalytic residue for the ꢀ-ketoacyl
synthase (KS) domain of CalE8,14 was replaced with alanine to
give CalE8-C211A, a KS° mutant of CalE8 that is colorless when
purified from E. coli (Figure 2B). The KS domain is responsible
for catalyzing the decarboxylative condensations of malonate units
necessary for polyketide chain extension. Accordingly, inactivation
of this domain affords a PKS that is incapable of polyketide
More recently, the methyl hexaenone 2, derived from the actions
of the calicheamicin PKS CalE8 and its downstream thioesterase
CalE7, was characterized.16 Liang and co-workers argued that a
divergence of the biosynthetic pathways to the 9- and 10-membered
enediynes begins at the PKS stage based on the apparent control
of oxidation states by each PKS during the last round of chain
extension. They also noted differences in the UV/vis spectra of
† Present address: Department of Biological Chemistry and Molecular Pharma-
cology, Harvard Medical School, Boston, Massachusetts 02115.
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C O M M U N I C A T I O N S
Figure 2. (A) UV/vis spectrum of the PKS CalE8 as purified from
heterologous expression in E. coli. Inset shows expansion from 305 to 480
nm. (B) Colorless CalE8-C211A (KS°) and yellow CalE8 (E8) bound to
Ni-NTA resin prior to elution.
Figure 4. Heptaene 1 is synthesized from malonyl-CoA by CalE8 and
CalE7, along with octaketides 2 and 3. Left panel: HPLC chromatogram
of a typical in vitro reaction, recorded at 375 nm. Right panel: Absorption
spectrum of the HPLC peak at 24 min showing the signature fine structure
of a hydrocarbon heptaene.
synthesis, providing an ideal control for our investigations. This
mutant also proved essential for optimization of sample preparation
and LC/MS analysis of the frequently labile reaction products.
The methyl ketone 2 characterized by Liang and co-workers was
observed early in our in Vitro studies of calicheamicin biosynthetic
enzymes as well (see Supporting Information). We have additionally
been able to identify its anticipated precursor, 3-oxohexadeca-
4,6,8,10,12,14-hexaenoic acid (3), in extracts from the same in Vitro
reaction of CalE8 and CalE7. A broad peak in the HPLC
chromatogram was found to have an exact mass of m/z ) 259.1373
(MH+), providing the first direct evidence for this presumed
hydrolysis product of CalE8. The decarboxylation of this labile
compound to 2 over time can be monitored by HPLC, further
supporting its assignment as the ꢀ-keto acid 3 (Figure 3). Although
this observation does not establish whether CalE7 is responsible
for catalyzing this decarboxylation as has been proposed,17 it does
prove that this transformation can proceed nonenzymatically.
In addition to corroborating the production of methyl ketone 2
and identifying its immediate precursor 3, we have observed
heptaene 1, the proposed common progenitor to 9-membered
enediynes, as a prominent product of the in Vitro reaction of CalE8
and CalE7, the biosynthetic enzymes for the 10-membered enediyne
calicheamicin. The distinctive UV/vis signature and HPLC retention
behavior in combination with exact mass confirmation identify the
peak at 24 min as heptaene 1 (Figure 4). The hydrocarbon heptaene
is unstable to a variety of conditions including light, heat, and
acidsbehavior that has been noted for the carotenoids and other
hydrocarbon polyenes.18 Liang and co-workers quenched their in
Vitro reactions with trifluoroacetic acid; this is likely why they were
not able to observe the sensitive heptaene.
of enediyne biosynthesis in which the highly similar PKS enzymes
from both 9- and 10-membered systems share a common blueprint
for polyketide assembly.19
The observed octaketide products 1-3 can be rationalized using
only the expected functions of CalE8 and CalE7, or any enediyne
PKS/thioesterase pair (Scheme 1). CalE8 and the enediyne PKSs
are predicted to have ꢀ-ketoreductase (KR) and dehydrase (DH)
domains, which catalyze the NADPH-dependent reduction of the
ꢀ-ketothioester intermediates and dehydration of the resulting
hydroxyl groups, respectively. Execution of these two processing
steps on successive rounds of ketide extension would lead to
polyene-conjugated ꢀ-ketothioesters represented by 4. If continued
until the full programmed chain length was reached, the product
of the final condensation would be enzyme-bound octaketide 5,
which contains a hexaene moiety. Using the thioesterase activity
of CalE7, ꢀ-ketothioester 5 could be released from CalE8 directly,
leading to detected products ꢀ-keto acid 3 and methyl ketone 2
(Via decarboxylation of 3). Alternatively, if the KR domain of CalE8
catalyzes one final reduction during this last round of chain
extension to give ꢀ-hydroxythioester 6, hydrolysis by CalE7 would
release hypothetical free acid 7. Dehydration and decarboxylation
would lead to heptaene 1, which has now been observed as another
major component of the in Vitro reaction of CalE8 and CalE7.
In accord with our own observations, CalE8 has also recently
been reported to produce a series of compounds having the
Scheme 1. Biosynthetic Rationale for the Observed Products
The discovery of heptaene 1, previously seen only in 9-membered
enediyne systems, as a major product of the calicheamicin enzymes
CalE8 and CalE7 brings a sharper perspective to enediyne bioge-
netic theory. It is the first experimental evidence indicating that
differentiation of immature polyketides to 9- or 10-membered
enediyne precursors is not solely inherent to the enediyne PKSs.
This observation vivifies the argument for a more convergent view
Figure 3. HPLC chromatogram of in Vitro reaction products of CalE8
and CalE7, recorded at 375 nm. Decarboxylation of ꢀ-keto acid 3 to methyl
ketone 2 occurs spontaneously over time in reaction extracts.
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C O M M U N I C A T I O N S
molecular formula CXHXO3 during in Vitro reactions.17 These have
been suggested to correspond to tetra-, penta-, and hexaketide
pyrone derailment products (8-10). These pyrones could arise from
offloading of PKS-bound intermediates similar to 4, whereby one
more round of chain extension and a simple cyclization would
release the 2-pyrones. In addition to these products, we also see
the simple triacetic acid lactone (11).
Facility at the University of Maryland, College Park and to Dr.
Alexei Gapeev, Director of Mass Spectrometry Facility at the
University of Maryland, Baltimore County for their assistance with
high and low resolution mass spectrometry, respectively. This work
was supported by NIH Grant ES001670. J.M.C. is currently a fellow
supported by the Damon Runyon Cancer Research Foundation
(DRG-2002-09, Harvard Medical School).
Iterative PKSs are generally regarded as highly processive,
releasing truncated or incorrectly processed polyketides in only trace
amounts unless denied an essential component of the synthetic
process.20,21 The simultaneous production of two different 15-
carbon linear polyenes by CalE8 with the assistance of thioesterase
CalE7 indicates that the calicheamicin PKS is not selectively
stopping at the ketone oxidation state in the final round of chain
extension. The synthesis of truncated polyketides 8-11 alongside
octaketides 1 and 2 leads us to suspect that CalE8 may be missing
some necessary biosynthetic element. Canonical nonribosomal
peptide synthetase (NRPS) and multidomainal PKS systems typi-
cally have all of the required modifying domains already encoded
in their sequences. There is, however, a growing body of literature
elucidating biosynthetic enzymes that act in trans on carrier protein-
tethered intermediates, often prior to full chain extension.22-24 The
absence of such accessory enzymes frequently results in biosynthetic
errors, which can manifest in a variety of ways.
In the landmark case of lovastatin biosynthesis, Hutchinson,
Vederas, and co-workers were able to show that without LovC, a
trans-acting enoyl reductase (ER), the lovastatin nonaketide syn-
thase LovB does not synthesize a full-length product. Instead, LovB
releases conjugated hexa- and heptaketide pyrone derailment
products,25 strikingly similar to the probable structures for CalE8
truncation products 8-10. A recent case from tenellin biosynthesis
describes the characterization of aberrant polyketide products that
reach the full programmed chain length but have incorrect oxidation
patterns when the mixed NRPS-PKS TenS is deprived of its
interaction with a trans-acting ER.26 The octaketide polyenes 1, 2,
and 3 produced by the calicheamicin biosynthetic enzymes may
also represent an instance of errant PKS behavior in the absence
of required auxiliary enzymes, despite the fact that they reflect the
full polyketide chain length expected for the calicheamicin aglycone.
We take the observations of both the methyl ketone 2 and the
heptaene 1 from in Vitro reactions of CalE8 and CalE7 as evidence
that neither is the branchpoint metabolite to 10-membered and
9-membered enediynes, respectively. The similar production of the
methyl ketone 2 from a 9-membered system, in addition to already-
observed heptaene 1, would bolster the argument for a more
convergent model of enediyne biosynthesis and support our
contention that divergence to 9- or 10-membered products results
from the action of one or more accessory enzymes acting in concert
with the enediyne PKS.
Supporting Information Available: Detailed experimental proce-
dures, HPLC conditions, and MS data for 1-3 and 8-11. This material
is available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. We are grateful to Dr. Yue Li, Director of
the Department of Chemistry and Biochemistry Mass Spectrometry
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