Modular polyketide synthases and cis double bond formation: Establishment of activated cis-3-cyclohexylpropenoic acid as the diketide intermediate in phoslactomycin biosynthesis

Article abstract of DOI:10.1021/ja068818t

The majority of modular polyketide synthase (PKS) systems which generate unsaturated products do so with trans double bonds. Phoslactomycin B (PLM B) presents a class of antitumor and antiviral natural polyketide products that have unique structural features, including a linear unsaturated backbone with one trans and three cis double bonds. There is substantial evidence that trans double bonds are established by ketoreductase-dehydratase (KR-DH) didomains within a PKS module. In cases where modules containing these didomains appear to generate product containing a cis double bond, there is no experimental evidence to determine if they do so directly or if they also form a trans double bond with a subsequent isomerization step. A critical step in addressing this issue is establishing the stereochemistry of the polyketide intermediate which passes to the subsequent module. Herein, we demonstrate through a series of experiments that an activated cis-3-cyclohexylpropenoic acid is the diketide intermediate which passes from module 1 to module 2 of the PLM PKS. The trans isomer of the diketide intermediate could not be processed directly into PLM B by module 2 but could be converted to PLM B by degradation to cyclohexanecarboxylic acid and elongation by the entire PLM PKS. These observations indicate not only that module 1 with a DH-KR didomain is responsible for establishing the C₁₄-C₁₅ cis double bond of PLM B but also that the subsequent modules of the PKS clearly discriminate between the cis- and trans-diketide intermediate and do not contain domains capable of catalyzing double bond isomerization. Copyright

Full text of DOI:10.1021/ja068818t

Published on Web 01/27/2007
Modular Polyketide Synthases and cis Double Bond Formation:
Establishment of Activated cis-3-Cyclohexylpropenoic Acid as the Diketide
Intermediate in Phoslactomycin Biosynthesis
Mamoun M. Alhamadsheh, Nadaraj Palaniappan, Suparna DasChouduri,‡ and Kevin A. Reynolds*
Department of Chemistry, Portland State UniVersity, Portland, Oregon 97207
Received December 15, 2006; E-mail: reynoldsk@pdx.edu
Phoslactomycins (PLMs), exemplified by PLM B (Figure 1), are
a unique class of antitumor, antiviral, and antifungal polyketide
natural products.1,2 The antitumor activity of PLMs is attributed to
a potent and selective inhibition of protein Ser/Thr phosphatase
2A (PP2A).3 The PLM biosynthetic gene cluster from Streptomyces
sp. HK803 has been cloned and sequenced.4 The PLM polyketide
synthase (PKS) is a modular PKS comprised of a loading domain
Figure 1. Phoslactomycin B (PLM B).
and seven extension modules which are responsible for the synthesis
of a unique linear unsaturated polyketide structure containing three
cis (Z) and one trans (E) double bonds.
starter unit. We generated a ∆chcA mutant (NP3), blocked in
biosynthesis of the starter unit, and demonstrated that it only
produces PLM B when grown in the presence of CHC (Table 1).
The trans- and cis-diketide products of PLM1 were synthesized in
both the acid (2a and 3a, Figure 2) and N-acetylcysteamine (SNAC)
thioester (4a and 5a, Figure 2) forms and added to separate
fermentations of this ∆chcA mutant. Surprisingly, compounds
2a-5a all restored PLM B production. PLM B production levels
were the highest for the trans products (2a and 4a) and were 40%
higher than that observed with either CHC supplementation or the
cis-SNAC (5a) (Table 1). The lowest level of PLM B production
was observed with the cis-acid (3a). Interestingly, the PLM B
isolated from feeding trans-acid 2a had the C14-C15 double bond
Modular PKSs which generate unsaturated products typically do
so using trans double bonds.5 These double bonds are established
by ketoreductase-dehydratase (KR-DH) domains which sequen-
tially carry out ketoreduction and dehydration steps on the 3-
ketoacyl-ACP products of the KS domains. The dehydration step
makes the stereochemical course of the KR-catalyzed step cryptic.
Recently, in vitro work using a DH-inactivated module 2 of the
pikromycin PKS, which establishes the single trans double bond
of pikromycin and methymycin, has shown this KR generates the
D-3-hydroxy product.6 A bioinformatic analysis of other cryptic
KR-DH domains which generate trans double bonds infers a
D-hydroxyl configuration (this analysis is based on an established
correlation of diagnostic residues in KR primary sequences and
their known stereochemical products).5,7
1
in the cis configuration, as confirmed by H NMR and NOESY
experiments. This initial result suggested that the trans-diketide
intermediate might be the preferred substrate for PLM2, with a
subsequent trans to cis isomerization step.
Polyketide products containing cis double bonds are rare and
appear to arise through a variety of mechanisms.8 In many cases,
such as modules 7 of PLM and module 4 of the epothilone PKS,
the required DH activity is absent from the module.4,9 Modules 1
and 2 of the PLM PKS are intriguing because they have combined
KR-DH didomains which appear to establish two conjugated cis
double bonds (C12-C13 and C14-C15 of PLM B, respectively).4
Bioinformatic analysis of the primary sequence of these KR
domains does not clearly predict a D-hydroxyl configuration (which
evidence indicates precedes trans double bond formation) or
L-hydroxy configuration (which has been speculated might precede
cis double bond formation).7 Thus, in each case, the combined
activity of these KR-DH didomains might establish a trans double
bond with a subsequent isomerization step to a cis double bond
(epimerization domains, in both PKS10 and NRPS11 modules, as
well as trans to cis double bond isomerization in retinoid cycle12
have been reported). Alternatively, these KR-DH domains might
establish the cis double bond directly.
Alternatively, the trans compounds might be converted efficiently
to the activated CHC starter unit by fatty acid degradation and
subsequently elongated by the entire PLM PKS (in this way, the
trans double bond would be lost through degradation and reintro-
duced as a cis double bond by PLM1) (Figure 2). To distinguish
between these two hypotheses, we synthesized and fed the [2-13C]-
labeled analogues 2b-5b (Figure 2) to the ∆chcA mutant. Mass
spectroscopy revealed that isotopic enrichment over natural abun-
dance for the PLM B product was only observed with the cis-SNAC
5b (20% isotope enrichment, Table 2). These data showed that both
cis and trans compounds undergo degradation to form the activated
CHC starter unit, and that this is the primary route for PLM B
production in these experiments. Furthermore, the experiments
established that only cis-SNAC (5a, 5b) could prime PLM2 directly.
The cis-acid (3a, 3b), which gives the lowest levels of PLM B
restoration levels, can be transported into the mutant and degraded
to the activated CHC (at about 50% the efficiency of the
corresponding trans-diketides) but cannot be activated intact such
that it can prime PLM2.
In this work, we have distinguished between these two possibili-
ties by determining the stereochemistry of the polyketide intermedi-
ate which is transferred from module 1 to module 2. PLM1 contains
a loading domain and the first extension module of the PKS and is
predicted to generate either cis- or trans-3-cyclohexylpropenoic acid
(Figure 2) from an activated cyclohexanecarboxylic acid (CHC)
A consistent and predictable set of results was obtained by
generation and analysis of a plm1 deletion mutant [NP9, see
Supporting Information] (Figure 2). PLM B production was
abrogated in this mutant and was only significantly restored by
growth in the presence of the cis-SNAC compounds 5a and its
13C-labeled counterpart 5b (Table 1). In the case of 5b, the PLM
B now contained the same level of isotopic enrichment (>99%) as
‡ Current address: Virginia Commonwealth University.
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10.1021/ja068818t CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Incorporation of CHC, compounds 2a-5a, and 2b-5b into PLM1 and PLM2 of PLM B PKS.
Table 1. Relative % of PLM B Production by Feeding CHC and
Compounds 2a-5a to ∆chcA and ∆plm1 Mutants
degradation to an activated CHC and elongation using PLM1. The
product of PLM1 must therefore be the cis-3-cyclohexylpropenoic
acid. These experiments also demonstrate that the PLM biosynthetic
process cannot process the trans-diketide intermediate either into
PLM B (ruling out an isomerization domain in the subsequent PKS
modules) or a PLM analogue with trans C14-C15 double bond. This
last observation indicates significant challenges to successful
alteration of the stereochemistry of unsaturated polyketide products
through either directed biosynthesis or KR-DH didomain switches.
∆chcA
∆plm1
substrate
mutant
mutant
control
CHC
2a
3a
4a
0
0
0
0
0
68 ( 3.9
100 ( 7
50 ( 3
98 ( 6
72 ( 7
∼0.5a
5a
100
Acknowledgment. Funding for this research was generously
provided by the National Institutes of Health (AI51629).
a LC-MS analysis demonstrated that 4a contained trace levels of 5a
(<1%).
Table 2. Percent of 13C Isotope Enrichment in Produced PLM B
Generated by Feeding CHC and Compounds 2b-5b to ∆chcA
and ∆plm1 Mutants
Supporting Information Available: Experimental procedures and
spectroscopic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
∆chcA
∆plm1
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