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experiments revealed higher relative incorporation efficiency at
lower concentration of the respective malonic acid derivative
toxic effects on K-Ras-dependent human pancreatic tumor
[13]
cells. Premonensin B was found to bind to PDEd in a compet-
itive fluorescence polarization assay with farnesylated Rheb
peptide, which has been used as a mimic of the K-Ras C-termi-
(Section II.2 in the Supporting Information). Higher concentra-
tions were increasingly toxic to the bacterium. Addition of
propyl-malonyl-SNAC nearly abolished the production of pre-
monensin and resulted in only trace amounts of the natural
product derivative. Interestingly, the relative amounts of the
naturally produced products premonensins A and B appeared
to be inverted in the LC-ESI-MS analysis upon addition of the
artificial building blocks.
nal peptide (K =(214ꢀ10) nm, see Figure 4 and Section III in
D
For propargyl-MSNAC, the feeding experiment was scaled
up to 1.8 L, as its preparative incorporation might give rise to
a synthetically useful orthogonal functional group in the poly-
ketide (Section II.4 in the Supporting Information). After purifi-
cation, the identity of propargyl-premonensin was confirmed
1
13
by H- and C NMR (Figure 3D and Section II.5 in the Support-
ing Information), thus proving specific incorporation of the
building block by AT5 . The new product was isolated in
mon
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1
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a
1
yield of 0.55 mgL
(5.4 mgL
premonensin A and
Figure 4. A 1 mm solution of fluorescently labeled Rheb peptide in the pres-
ence of 1 mm PDEd was titrated against increasing concentrations of premo-
nensin B (2a) and propargyl-premonensin. Premonensin B showed an IC50 of
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1
.8 mgL premonensin B from the same fermentation). This
confirmed the inversion of the incorporation efficiencies of
ethyl- and methyl-malonyl-CoA upon addition of the artificial
malonic acid derivative (control fermentations yielded
0.81 mm whereas propargyl-premonensin (5a) showed an IC50 of 2.00 mm.
~
: negative control,
&
: premonensin B, and *: propargyl-premonensin.
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1
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8
.2 mgL premonensin A and 15.2 mgL premonensin B).
In further investigation of the substrate flexibility of the PKS,
the Supporting Information). Premonensin B was able to dis-
place the natural binder from the complex with PDEd. The
propargylated derivative showed a 2.5-fold decreased affinity
towards the protein (Figure 4 and Section III in the Supporting
Information).
all artificial building blocks except butylmalonyl-SNAC were
supplied to cultures of the mutant strain S. cinnamonensis
0
[9]
A495-ER2 . This strain gives rise to a redox derivative of pre-
monensin (Figure 3A) and was the result of previous PKS-engi-
[9]
neering experiments to yield polyketide redox derivatives.
This result shows that modified biosynthesis can produce
suitable starting compounds for natural-product based struc-
ture-activity studies. In this context, the newly attached prop-
argyl moiety will, after further improvement of this precursor-
directed biosynthesis experiment, offer opportunities for
straightforward chemical modification in a chemistry–biology–
Analysis of these fermentations revealed interesting differences
from the wild-type PKS and indicates “crosstalk” between dif-
ferent segments of the multienzyme complex by unknown
mechanisms. As expected, the overall amounts of the poly-
ketide derivatives were significantly lower than for the wild-
type PKS. Interestingly, however, propargyl-MSNAC did not
give rise to the expected premonensin derivative but instead
largely abolished productivity. Previously, a small amount of
a biosynthetic byproduct corresponding to the incorporation
[14]
chemistry reaction sequence.
Conclusion
[
9]
of MCoA by AT5mon was isolated. Propyl- and allyl-malonyl-
SNAC on the other hand led to significant incorporation levels
relative to wild type. These experiments indicate a subtle but
effective crosstalk mechanism between module 2 and
module 5. Apparently, the differences in the surrounding PKS
machinery can induce alterations of the substrate specificity of
individual domains or modules. This could be the result of
dedicated proofreading mechanisms or simply substrate-in-
Here we present the first computational model of the AT5mon
domain of the monensin PKS; this domain plays an essential
role in building-block selection and incorporation. This model
allowed us to predict the incorporation of non-native sub-
strates in the wild-type system and to investigate the basis of
substrate recognition by this enzyme. Our predictions were
corroborated and extended by systematic screening for the in-
corporation of non-natural malonic acid derivatives as building
blocks for this PKS. Important findings are that the inclusion of
artificial building blocks seemed to reverse the relative
amounts of the shunt products premonensin A and B. More-
over, premonensin B and propargyl-premonensin (obtained
after theoretically predicting the incorporation of propargyl-
MSNAC by wild-type AT5mon) bound to PDEd. The incorporation
of the new propargyl moiety into the structure of premonensin
opens perspectives for the discovery of further bioactive deriv-
atives through targeted biosynthetic derivatization and chemi-
cal conversions.
[
9,11]
duced alteration of catalytic activity.
Bioactivity of premonensin and its derivative
Polyketides have specific chemical structures to interact with
[
12]
biological macromolecules. Here we found that the non-nat-
ural biosynthetic shunt-product premonensin binds tightly to
the human phosphopdiesterase 6 delta subunit (PDEd). This
protein plays a pivotal role in K-Ras trafficking in human cells,
and its inhibition by suitable binders was shown to have cyto-
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
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