Predicted incorporation of non-native substrates by a polyketide synthase yields bioactive natural product derivatives

Article abstract of DOI:10.1002/cbic.201402206

The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl- or methyl-malonyl-CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5_mon) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5_mon, insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non-native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl-binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules. PKS analysis: Which substrate is incorporated, how does it happen and what is produced? Computational modeling of the fifth acyltransferase domain of a polyketide synthase predicts that the enzyme incorporates non-native building blocks into the polyether ionophore monensin to give natural product derivatives with biological activity.

Full text of DOI:10.1002/cbic.201402206

CHEMBIOCHEM
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DOI: 10.1002/cbic.201402206
Predicted Incorporation of Non-native Substrates by
a Polyketide Synthase Yields Bioactive Natural Product
Derivatives
[
a]
[b]
[b]
[b]
Kenny Bravo-Rodriguez, Ahmed F. Ismail-Ali, Stephan Klopries, Susanna Kushnir,
[c, d]
[c]
[c]
[b]
Shehab Ismail,
Eyad K. Fansa, Alfred Wittinghofer, Frank Schulz,* and Elsa Sanchez-
[a]
Garcia*
The polyether ionophore monensin is biosynthesized by a poly-
ketide synthase that delivers a mixture of monensins A and B
by the incorporation of ethyl- or methyl-malonyl-CoA at its
fifth module. Here we present the first computational model of
the fifth acyltransferase domain (AT5mon) of this polyketide syn-
thase, thus affording an investigation of the basis of the re-
laxed specificity in AT5mon, insights into the activation for the
nucleophilic attack on the substrate, and prediction of the in-
corporation of synthetic malonic acid building blocks by this
enzyme. Our predictions are supported by experimental stud-
ies, including the isolation of a predicted derivative of the
monensin precursor premonensin. The incorporation of non-
native building blocks was found to alter the ratio of premo-
nensins A and B. The bioactivity of the natural product deriva-
tives was investigated and revealed binding to prenyl-binding
protein. We thus show the potential of engineered biosynthet-
ic polyketides as a source of ligands for biological macromole-
cules.
Introduction
Polyketide synthases (PKSs) are natural multienzyme com-
plexes of high relevance for medicine as they synthesize com-
pounds used as immunosuppressants, antibiotics, and anti-
cancer drugs, among others. PKSs catalyze a cascade of Claisen
condensations between enzyme-bound acyl thioesters and ma-
lonic acid thioesters, which serve as extender units for the
growing polyketide chain. In bacterial type I PKSs, acyltransfer-
ase (AT) domains serve as gatekeepers to recruit the malonic-
acid-based building blocks required for chain extension. The
mechanism of this process is largely not understood, thus ren-
dering the task of engineering a PKS for the incorporation of
substrate incorporation into PKS is a task of high importance,
but this is currently hindered by the lack of structural informa-
tion on the megadalton-sized PKSs.
The AT domain in module five of the monensin PKS (AT5mon
)
is known to accept both ethyl-malonyl- and methyl-malonyl-
CoA (EtMCoA and MMCoA) as substrates, thereby giving rise
[7,8]
to monensins A and B.
Previous mutagenesis experiments
have shown the flexibility of this PKS in processing non-native
substrates with different redox patterns in the nascent poly-
ketide chain, thus implying opportunities for further biosyn-
[9]
thetic derivatization of monensin. Furthermore, our earlier in-
vestigations on artificially induced substrate promiscuity in the
[
1–6]
non-native building blocks a formidable challenge.
In this
context, elucidation of the molecular mechanisms underlying
sixth AT domain of the erythromycin assembly line (AT6DEBS)
showed that a non-natural building block can be introduced
as a source of a propargylated side chain in erythromycin A by
+
[
a] K. Bravo-Rodriguez, Dr. E. Sanchez-Garcia
[5]
targeted mutagenesis of the AT6DEBS domain. However, it is
important to broaden and deepen the understanding of sub-
strate recognition by giant and complex PKSs, in particular of
the AT domains, as these are crucial for the introduction of
new functional groups and stereocenters.
Department of Theoretical Chemistry
Max-Planck-Institut fꢀr Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 Mꢀlheim an der Ruhr (Germany)
E-mail: esanchez@mpi-muelheim.mpg.de
+
[
b] A. F. Ismail-Ali, S. Klopries, Dr. S. Kushnir, Prof. Dr. F. Schulz
Fakultꢁt fꢀr Chemie und Biochemie, Ruhr-Universitꢁt Bochum
Universitꢁtsstrasse 150, 44780 Bochum (Germany)
E-mail: frank.schulz@rub.de
[
c] Dr. S. Ismail, E. K. Fansa, Prof. Dr. A. Wittinghofer
Max-Planck-Institut fꢀr Molekulare Physiologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Results and Discussion
Molecular modeling of acyltransferase domain 5 of monen-
sin PKS
[d] Dr. S. Ismail
Present address: CR-UK Beatson Institute, Garscube Estate
Switchback Road,Glasgow G61 1BD (UK)
We built a homology model of the AT5mon domain in order to
investigate its substrate recognition mechanism (see Computa-
tional details and the Supporting Information). Based on the
amino acid sequence of AT5mon, we used the X-ray structures
+
[
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402206.
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of AT3DEBS, AT5DEBS, and malonyl CoA/acyl carrier protein transa-
cylase (MAT) as templates (Section I.1 in the Supporting Infor-
mation). After equilibration, we performed molecular dynamics
(MD) simulations of solvated AT5mon without ligand. These sim-
ulations indicated that the overall protein structure is con-
served. However, in the absence of substrate, the region
formed by residues 267–339 was most flexible, as it tended to
move away from the rest of the protein. This movement
makes the active site of AT5mon more open and exposed to the
ligand than in AT6DEBS
.
MD simulations of AT5mon models with the natural substrates
MMCoA or EtMCoA) in different orientations showed, in all
(
cases, a structured active site able to accommodate the li-
gands. We found that the main interaction between MMCoA
and the active site is the salt bridge formed between the car-
boxylate group of MMCoA and R257, which is located at the
bottom of the active site. The thioester and amide groups of
MMCoA further contribute to keeping the ligand in a suitable
position within the active site (Section I.2 in the Supporting In-
formation). The simultaneous interactions of the thioester
moiety and the closest amide group of MMCoA with the back-
bone of Q149 help the substrate to adopt a conformation in
which S232 is close to the thioester, thus enabling the nucleo-
philic attack (Figure 1A). Our work also provides insights into
the activation of S232. In all simulations, S232 interacted with
H231 (Section I.2 and Table S1 in the Supporting Information)
or with the carboxylate group of the ligand, but never with
H334. This suggests that either H231 or the carboxylate group
Figure 1. A) Residue S232 interacts with H231 but not with H334. B) A com-
parison of AT5mon with AT6DEBS (italics) indicates that the VAGH sequence and
S329 might play a role in the substrate promiscuity of the monensin PKS.
(but not H334) acts as the base to promote activation of S232
in AT5mon
.
YASH is VAGH, and S329 is the residue equivalent to V295 (Fig-
ure 1B). Our simulations indicated that these differences result
in a more open active site that is thus able to accept not only
MMCoA but also EtMCoA as a substrate. EtMCoA establishes
an interactions network with the active site similarly to
MMCoA. The most favorable orientation of this substrate is
with the ethyl group pointing towards V331 (Figure 2A).
Unlike MMCoA, EtMCoA was found in a location distant from
S232 if the ethyl moiety was not in the correct orientation
(Section I.2 in the Supporting Information). In all cases, the in-
By incorporating EtMCoA, AT5mon exhibits a relaxed specifici-
ty compared to AT6DEBS, which is highly selective for MMCoA. A
[
5]
comparison of AT5mon with our AT6DEBS model provides inter-
esting insights into possible factors contributing to substrate
promiscuity in AT5mon. In AT6DEBS, the YASH sequence strongly
interacts with the substrate and together with V295 prevents
the incorporation of substrates larger than MMCoA in the
active site. We established that the V295A mutation lowers the
[
5]
specificity of AT6DEBS
.
In AT5mon, the sequence equivalent to
Figure 2. Binding at the active site of AT5mon by A) EtMCoA (native substrate), B) MCoA, and C) propargyl-MSNAC. The substrates are shown as thicker stick
models. Unlike EtMCoA, MCoA lacks many stabilizing interactions with the active site. Propargyl-MSNAC is accommodated in the active site in an orientation
similar to that of EtMCoA.
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teraction with R257 was conserved, and thus EtMCoA re-
mained inside the active site.
Experimental investigation of substrate promiscuity of
AT5mon
Given the experimental complexity of PKS manipulation, the
ability to computationally predict the incorporation of non-
native substrates is important, not only as a guide to experi-
ments but also to understand the fundamental substrate selec-
tivity of PKS. The relaxed specificity of AT5mon relative to
AT6DEBS and other AT domains does not automatically establish
AT5mon as a very promiscuous enzyme, as MMCoA and EtMCoA
differ by only a methylene group. In this context, we wanted
to establish whether AT5mon is able to incorporate synthetic
building blocks with larger and more rigid moieties than
a methyl or ethyl group.
Malonic acid derivatives, each thioester-activated with N-acetyl-
cysteamine (SNAC), were synthesized either from commercially
available derivatives of malonic acid diesters or by reductive al-
kylation of Meldrum’s acid (Scheme 1, Figure 3A; and Section
[10]
IV in the Supporting Information).
Thus, we computationally investigated the incorporation of
two ligands of different sizes relative to MMCoA: the smaller
natural malonyl-CoA (MCoA) and the larger synthetic proparg-
yl-malonyl-SNAC (propargyl-MSNAC; Figure 2B and C).
Simulations of AT5mon with bound MCoA suggested signifi-
cantly weakened interaction with the active site: average
values of the distance between R257 and the carboxylate
moiety of MCoA were large, the substrate was far from S232,
and no secondary stabilizing interactions between the ligand
and the active site were found (Figure 2B; Section I.2, and
Table S1 in the Supporting Information). This is consistent with
experimental observations: in premonensin and monensin bio-
synthesis, the abundantly available natural MCoA does not
lead to the corresponding desmethyl derivatives of the natural
[
9]
products in significant quantities.
Propargyl-MSNAC, on the other hand, was significantly
better accepted by the active site and exhibited a behavior
similar to that of the natural substrate, EtMCoA. However, un-
favorable orientations of this ligand led to disruption of several
interactions important for substrate binding and activation
(with residues such as Q149, Q233, and R257; Section I.2 in the
Supporting Information). Our results thus indicate that the in-
sertion of propargyl-MSNAC into the active site depends on
the orientation of its triple-bond moiety, which should point
toward V331 similarly to the ethyl group in EtMCoA (Figure 2A
and C).
Scheme 1. Synthesis of compounds 4–8; a 3.0 equiv LiOH·H
2 2
O, H O, 18 h,
RT; b) 1.01 equiv isoprenylacetate, 0.06 equiv H SO , neat, 18 h, RT;
2
4
c) 1.0 equiv borane dimethylamine complex, 3.0 equiv propyl, butyl, or hexyl
In addition to homology modeling and MD simulations we
performed quantum mechanics/molecular mechanics (BP86-
D2/SVP//CHARMM) calculations of the AT5mon–propargyl-
MSNAC system; the results were in very good agreement with
the MD results (Supporting Information, Section I.2 and
Table S5). The relative free energy differences associated with
the transformations MMCoA!EtMCoA and MMCoA!proparg-
yl-MCoA were also calculated. To directly compare the effect of
the substituents, we calculated propargyl-MCoA instead of
propargyl-MSNAC, which was used in the experiments. As
aldehyde, 1 h, RT; d) tBuOH, 6 h, 90–1008C; e) 1.1 equiv CDI, 0.3 equiv DMAP,
4
1.2 equiv SNAC, THF, 18 h, RT; f) 2.5 equiv TiCl , DCM, 6 h, RT. CDI: N,N’-Car-
bonyldiimidazole, DMAP: 4-dimethylaminopyridine, SNAC: N-acetylcystea-
mine.
Cultures of Streptomyces cinnamonensis A495 were supple-
mented with each derivative (10–30 mm). This strain produces
premonensin, an artificial shunt-product of the monensin bio-
[8]
synthetic pathway. Analysis of the fermentation extracts by
LC-ESI-MS and HRMS indeed revealed significant PKS promiscu-
ity (Section II.3 in the Supporting Information). Four of the five
synthetic building blocks were incorporated in significant
amounts. 2-Hexylmalonyl-SNAC was found to be toxic for the
bacterium and to abolish the fermentation of premonensin.
LC-ESI-MS analysis showed that each new premonensin deriva-
tive was as a third fermentation product, in addition to premo-
nensins A and B. This indicates specific incorporation of the
building blocks by the AT5mon domain. As a general trend, the
both MMCoA and EtMCoA are natural substrates of AT5mon
,
DDG should have values close to 0. As expected, the calculat-
ed value for the MMCoA!EtMCoA transformation was 0.12ꢀ
ꢁ
1
0
.02 kcalmol . The small DDG value for MMCoA!propargyl-
ꢁ1
MCoA (0.53ꢀ0.04 kcalmol ) means that, thermodynamically,
the active site of AT5 should be able to accommodate the
mon
bulky propargyl group as it does with the natural substrates.
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Figure 3. A) Malonic acid derivatives used in this study (those investigated by molecular modelling are in bold letters). From the synthetic building blocks, an-
alogues 4–7 were experimentally incorporated into premonensin. Compound 5a was isolated from the fermentation broth and analyzed by NMR. The corre-
0
sponding propargylated ER2 -derivative, however, was not found by LC-ESI-MS. B) LC-ESI-MS total ion chromatogram of a crude fermentation extract of S. cin-
+
+
+
namonensis A495 supplemented with 5. C) High-resolution MS results from the same fermentation extract; 5a is identified by its H , NH
4
, and Na adducts.
1
D) A segment of the H NMR analysis of premonensin A (3a, lower trace) in comparison to 5a (upper trace).
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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
ꢁ
1
ꢁ1
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
ꢁ
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.
ꢁ
1
ꢁ1
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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emission wavelengths of 495 nm and 520 nm, respectively, were
used. Data analysis was performed with Grafit 5.0 (Erithacus Soft-
ware, East Grinstead, UK).
Experimental Section
[34]
Computational details: Based on its amino acid sequence, the ho-
mology model of the AT5 domain of the monensin PKS (AT5mon
was generated from the X-ray structures of DEBS domains AT3
)
Stock solutions of premonensin B (10 mm, 2a) and propargyl-pre-
monensin (10 mm, 5a) were prepared in Milli-Q water/DMSO
DEBS
[15]
[16]
(
PDB ID: 2QO3) and AT5DEBS (PDB ID: 2HG4) and of malonyl-
(
(
20:80, v/v) supplemented with CAVASOL W7 HP Pharma
16.8 mgmL ; Applichem) and methanol (5 mL to pre-dissolve the
CoA:acyl carrier protein transacylase (MAT, to be published) as tem-
ꢁ1
[17]
plates. The program Modeller (v. 9.10) was used.
compound before dilution into DMSO/H O). Equivalent concentra-
2
[18]
The program NAMD2.9 was used for all MD simulations with the
tions of Milli-Q water and DMSO supplemented with methanol and
[
19]
[20]
ꢁ1
CHARMM22 force field for the protein and the TIP3P model
for water. Parameters for the substrates were generated by Swiss-
CAVASOL W7 HP Pharma (16.8 mgmL ) served as negative control.
More details of the experimental and computational procedures
are in the Supporting Information.
[21]
[5]
param and previously tested by us. The PME method was used
for the treatment of the electrostatic interactions.
[22]
[23–25]
QM/MM (BP86-D2/SVP//CHARMM22)
optimizations were also
used to study the interaction between propargyl-MSNAC and the
active site. Snapshots taken at the beginning and end of the simu-
lations were optimized and compared for an additional assessment
of the evolution of key contacts between the substrate and resi-
dues in the active site. The QM regions were defined as the entire
substrate. All residues within 30 ꢁ of the substrate were allowed to
move freely during the optimizations. The HDLC optimizer was
Acknowledgements
E.S.-G. acknowledges a Liebig-stipend from the Fonds der Chemi-
schen Industrieand the support of the SFB1093 funded by the
Deutsche Forschungsgemeinschaft. A F.I.-A. is a fellow of the In-
ternational Max Planck Research School of Chemical Biology.
This work was supported by the Cluster of Excellence RESOLV
[26]
[27]
used.
QM calculations were handled with Turbomole5.10;
[28]
DL_POLY was used for the MM calculations. All QM/MM optimi-
(
EXC 1069) funded by the Deutsche Forschungsgemeinschaft.
[29]
zations were performed with Chemshell3.5.
Relative free energy differences (DDG) were calculated to assess
the preference of the active site for different lateral chains of the
substrates. The transformations of MMCoA in EtMCoA, and prop-
argyl-MCoA were computed by using Free Energy Perturbation
Keywords: computational
calculations · molecular dynamics · monensin · polyketide
biosynthesis · QM/MM
chemistry
·
free
energy
[30,31]
theory.
parseFEP plugin of VMD. The Bennet acceptance ratio was used
The free energy calculations were analyzed with the
[32]
[33]
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to calculate the error in DDG.
[8]
Experimental details: S. cinnamonensis A495 (BHDCIBIBII) and
0
[9]
A495-ER2 was grown in tryptic soy broth (Fluka Analytical) at
ꢁ
1
ꢁ1
3
08C. SM16 medium (MOPS (20.9 gL ), l-proline (10 gL ), glu-
1
ꢁ
ꢁ1
ꢁ1
cose (20 gL ), NaCl (0.5 gL ), K2HPO4 (2.10 gL ), EDTA
0.25 gL ), MgSO ·7H O (0.49 gL ), CaCl ·2H O (0.029 gL )), sup-
4 2 2 2
ꢁ
1
ꢁ1
ꢁ1
(
plemented with the respective malonyl-SNAC derivative (10, 20, or
0 mm) was inoculated with pre-culture (5%). SM16 cultures were
grown for five days at 308C. On days 4 and 5, SM16 cultures were
[
6] I. Koryakina, J. McArthur, S. Randall, M. M. Draelos, E. M. Musiol, D. C.
Muddiman, T. Weber, G. J. Williams, ACS Chem. Biol. 2013, 8, 200–208.
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Wilkinson, Z. Oliynyk, Y. Demydchuk, J. Staunton, P. F. Leadlay, Mol. Mi-
crobiol. 2003, 49, 1179–1190.
3
[
ꢁ1
supplemented with XAD16 resin (20 gL , Sigma–Aldrich). S. cinna-
0
monensis A495 and A495-ER2 were also cultured without deriva-
tive as a control.
[8] A. Bhatt, C. B. W. Stark, B. M. Harvey, A. R. Gallimore, Y. A. Demydchuk,
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Published online on && &&, 0000
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
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These are not the final page numbers!

FULL PAPERS
K. Bravo-Rodriguez, A. F. Ismail-Ali,
S. Klopries, S. Kushnir, S. Ismail,
E. K. Fansa, A. Wittinghofer, F. Schulz,*
E. Sanchez-Garcia*
PKS analysis: Which substrate is incor-
porated, how does it happen and what
is produced? Computational modeling
of the fifth acyltransferase domain of
a polyketide synthase predicts that the
enzyme incorporates non-native build-
ing blocks into the polyether ionophore
monensin to give natural product deriv-
atives with biological activity.
&& – &&
Predicted Incorporation of Non-native
Substrates by a Polyketide Synthase
Yields Bioactive Natural Product
Derivatives
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
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8
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These are not the final page numbers!