Expanding the structural diversity of polyketides by exploring the cofactor tolerance of an inline methyltransferase domain

Article abstract of DOI:10.1021/ol401723h

A strategy for introducing structural diversity into polyketides by exploiting the promiscuity of an in-line methyltransferase domain in a multidomain polyketide synthase is reported. In vitro investigations using the highly-reducing fungal polyketide synthase CazF revealed that its methyltransferase domain accepts the nonnatural cofactor propargylic Se-adenosyl-l-methionine and can transfer the propargyl moiety onto its growing polyketide chain. This propargylated polyketide product can then be further chain-extended and cyclized to form propargyl-α pyrone or be processed fully into the alkyne-containing 4′-propargyl-chaetoviridin A.

Full text of DOI:10.1021/ol401723h

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Expanding the Structural Diversity of
Polyketides by Exploring the Cofactor
Tolerance of an Inline Methyltransferase
Domain
Jaclyn M. Winter,† Grace Chiou,‡ Ian R. Bothwell,§,^ Wei Xu,† Neil K. Garg,‡ Minkui Luo,^
and Yi Tang*,†,‡
Department of Chemical and Biomolecular Engineering and Department of Chemistry and
Biochemistry, University of California, Los Angeles, California 90095, United States, and
Tri-Institutional Training Program in Chemical Biology and Molecular Pharmacology
and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York
10065, United States
yitang@ucla.edu
Received June 18, 2013
ABSTRACT
A strategy for introducing structural diversity into polyketides by exploiting the promiscuity of an in-line methyltransferase domain in a multidomain
polyketide synthase is reported. Invitro investigations using the highly-reducing fungal polyketide synthase CazF revealed thatits methyltransferase
domain accepts the nonnatural cofactor propargylic Se-adenosyl-L-methionine and can transfer the propargyl moiety onto its growing polyketide
chain. This propargylated polyketide product can then be further chain-extended and cyclized to form propargyl-R pyrone or be processed fully into
the alkyne-containing 40-propargyl-chaetoviridin A.
S-Adenosyl-L-methionine (SAM, 1)-dependent methyl-
transferases are a highly versatile class of enzymes that
catalyze the methylation of DNA, RNA, protein, and
small molecules using the sulfonium methyl group of 1.1
Remarkably, these enzymes have been shown to accept
synthetic SAM analogues and can catalyze the transalk-
ylation of their corresponding protein,2 nucleic acid,3 and
small molecule4 substrates with reactive bioorthogonal
functional groups. In addition to freestanding methyl-
transferases, methyltranserase (MT) domains can also be
found within polyketide synthases (PKS) where they cat-
alyze the R-methylation of a growing β-keto polyketide
chain using 1 as the methyl-donating cofactor.5
(2) (a) Peters, W.; Willnow, S.; Duisken, M.; Kleine, H.; Macherey,
€
T.; Duncan, K. E.; Litchfield, D. W.; Luscher, B.; Weinhold, E. Angew.
Chem., Int. Ed. 2010, 49, 5170. (b) Islam, K.; Zheng, W.; Yu, H.; Deng,
H.; Luo, M. ACS Chem. Biol. 2011, 6, 679. (c) Wang, R.; Zheng, W.; Yu,
H.; Deng, H.; Luo, M. J. Am. Chem. Soc. 2011, 133, 7648. (d) Bothwell,
I. R.; Islam, K.; Chen, Y.; Zheng, W.; Blum, G.; Deng, H.; Luo, M. J.
Am. Chem. Soc. 2012, 134, 14905. (e) Binda, O.; Boyce, M.; Rush, J. S.;
Palaniappan, K. K.; Bertozzi, C. R.; Gozani, O. ChemBioChem 2011, 12,
330. (f) Wang, R.; Islam, K.; Liu, Y.; Zheng, W.; Tang, H.; Lailler, N.;
Blum, G.; Deng, H.; Luo, M. J. Am. Chem. Soc. 2013, 135, 1048. (g)
Blum, G.; Bothwell, I. R.; Islam, K.; Luo, M. Curr. Prot. Chem. Biol.
2011, 7, 2970. (h) Willnow, S.; Martin, M.; Luscher, B.; Weinhold, E.
ChemBioChem. 2012, 13, 1167.
† Department of Chemical and Biomolecular Engineering, UCLA.
‡ Department of Chemistry and Biochemistry, UCLA.
§ Tri-Institutional Training Program in Chemical Biology, Memorial
Sloan-Kettering Cancer Center.
^ Molecular Pharmacology and Chemistry Program, Memorial Sloan-
Kettering Cancer Center.
(1) Struck, A. W.; Thompson, M. L.; Wong, L.; Micklefield, J.
ChemBioChem. 2012, 13, 2642.
r
10.1021/ol401723h
XXXX American Chemical Society

Polyketide natural products are a structurally diverse
class of molecules isolated from bacteria, fungi, and plants
and represent a rich source of medicinally important
compounds.6 The complex structuresassociatedwithpoly-
ketides arise from the utilization of simple building blocks,
such as malonyl-CoA, and can undergo varying degrees of
β-keto reduction during each catalytic cycle on the PKS.7
As a result, enzyme-directed engineering approaches have
been aimed at diversifying building block and starter unit
selection as a way to enhance the structural variation of
polyketides and to create analogues containing nonnative
functional groups.8 Recently, enzyme engineering efforts
with bacterial acyltransferase (AT) domains9 and acyl-
CoA synthetases10 have been successful at assimilating
nonnatural moieties into polyketides using synthetic malo-
nic acid building blocks. While these synthetic biology
strategies are promising for enhancing the structural di-
versity of polyketides, an alternative method for introdu-
cing structural variation can involve the use of nonnatural
cofactors. Thus, given the reported promiscuous nature of
standalone methyltransferases, we wanted to investigate
whether MT domains found within PKSs could behave in
the same manner and facilitate the incorporation of dif-
ferent alkyl groups at the R-positions of polyketides.
To examine the substrate specificity of a PKS MT
domain, we turned our attention to the fungal highly-
reducing PKS (HR-PKS) CazF, which is involved in the
biosynthesis of the chaetoviridin and chaetomugilin
azaphilones.11 In vitro reconstitution experiments with re-
combinant CazF (284 kDa) expressed from Saccharomyces
cerevisiae BJ5464-NpgA12 verified that the MT domain
functions after the first chain-extending condensation step,
and the remaining domains are all catalytically active to
generate a small polyketide product.11 CazF therefore repre-
sented a good model system for exploring whether a MT
domain can be used to introduce chemical diversity into
polyketides. We selected the two unnatural SAM analogues
propargylic Se-adenosyl-L-methionine (ProSeAM, 2)2d,h and
keto-SAM (3)2d,13 for this study because both analogues can
provide a reactive handle that can be further modified with
mild, orthogonal chemistry. The selenium containing 2 was
selected over the sulfur-based propargylic SAM derivative
because it is more stable at physiological pH with a half-life of
approximately one hour.2d
Scheme 1. CazF-Mediated Transalkylation of 4 Using Its Natural
Cofactor 1 or Unnatural Analogues 2 or 3 as the Alkyl Donor
We first assayed the kinetics of the CazF MT domain
toward the unnatural cofactors. Recombinant CazF was
incubated with acetoacetyl-SꢀN-acetyl cysteamine
(acetoacetyl-SNAC, 4) in the presence of 1, 2, or 3
(Scheme 1). The production of alkylated acetoacetyl-SNAC
products were analyzed by liquid chromatography and mass
spectroscopy (LC-MS); and kinetic constants were calculated
by fitting initial velocity data at various concentrations of 1or
2 to MichaelisꢀMenten parameters using nonlinear least-
squares curve fitting. In the assays containing 3, no product
was detected suggesting that the MT domain would not
accept the ketone derivative. The alkylated 5 and 6 were
formed in the presence of 1 and 2, respectively. The steady-
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Biomol. 2011, 2, 211. (b) Winter, J. M.; Tang, Y. Curr. Opin. Biotech.
2012, 5, 736. (c) Wilson, M. C.; Moore, B. S. Nat. Prod. Rep. 2012, 29, 72.
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state kinetic parameters were kcat = 0.025 minꢀ1 and KM
=
15.5 μM for1and kcat = 0.036 minꢀ1 and KM = 43.6 μMfor
2. Although the turnover rates were slow for both 1and 2, the
comparable kinetic parameters indicate that the CazF MT
displays similar preference toward 1 and 2. The slow turn-
over rate may be attributed to the use of a small molecule
SNAC-bound substrate instead of an acyl-carrier protein
(ACP)-tethered molecule.
(11) Winter, J. M.; Sato, M.; Sugimoto, S.; Chiou, G.; Garg, N. K.;
Tang, Y.; Watanabe, K. J. Am. Chem. Soc. 2012, 134, 17900.
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B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 2. Synthesis of 9 Using 2,4-Pentanedione and
(3-Bromoprop-1-yn-1-yl)trimethylsilane15
Figure 1. R-Pyrones 7ꢀ9 biosynthesized by CazF in the presence
of (i) malonyl-CoA and SAM 1 or (ii) malonyl-CoA and
ProSeAM 2. HPLC traces are shown in the same scale at λ =
280 nm. Domain organization of CazF consists of a keto-
synthase (KS), malonyl-CoA:ACP acyltransferase (MAT), de-
hydratase (DH), methyltransferase (MT), enoyl reductase (ER),
ketoreductase (KR), and acyl carrier protein (ACP).
under the reaction conditions, the rate of MT-alkylation is
slower with the unnatural cofactor, and that the KS was
able to outcompete the MT domain in extending the chain
without alkylation. With the natural cofactor 1, MT-
catalyzed methylation is faster, which results in 8 as the
dominant product. Increasing the concentration of 2 to
1 mM did not alter the ratio of 9 and 7, which was expected
since the KM toward 2 was measured to be <50 μM.
Previous in vitro reconstitution experiments established
that when 1, along with NADPH used by the KR domain
are present, the triketide product 13 is synthesized by CazF
and is transacylated by the acyltransferase CazE to the
pyranoquinone intermediate cazisochromene (14) to form
chaetoviridin A (15) (Scheme 3).11 To further explore the
tolerance of PKS domains, such as that of the KR toward
the propargylated diketide and post-PKS accessory en-
zyme CazE toward the propargyl-containing polyketide
intermediate 16, we tested whether an alkyne-containing
analogue of 15, such as 17, can be synthesized using the
same set of enzymes. Equimolar amounts of CazF and
CazEwere incubated with 14, NADPH, malonyl-CoA and
2. LC-MS analysis of the organic extract revealed the
formation of a new product with a shorter retention time
than 15 and a [M þ H]þ m/z = 457 (Figure 2A) consistent
with that of 17. This new compound has an identical UV
profile to 15 and an isotopic mass ratio consistent with the
presence of one chlorine atom. Based on these observa-
tions, we assigned the product to be 40-propargyl-chaeto-
viridin A (17). To provide additional evidence toward the
identity of the new product, a Cu(I)-catalyzed azideꢀ
alkyne Huisgen cycloaddition reaction16 was performed
to verify if 17 did indeed contain a terminal alkyne moiety.
After reacting the organic extract containing 17 with azide-
PEG3-5(6)- carboxytetramethylrhodamine, formation of a
After confirming that the MT domain would accept 2
and transfer the propargyl moiety onto 4, we assessed
whether the β-ketoacyl synthase (KS) domain of CazF
would accept the propargylated diketide intermediate and
perform another decarboxylative Claisen condensation
reaction with malonyl-CoA to form the propargylated
triketide product. Previous in vitro characterization assays
with CazF demonstrated that the enzyme can produce the
triacetic acid lactone 7 when incubated with only malonyl-
CoA, and the dimethylated R-pyrone 8 when SAM was
included inthe reaction (Figure 1).11 To determine whether
CazF could produce a propargyl-R-lactone, the enzyme
was incubated with malonyl-CoA and 0.2 mM of 2.
Following incubation, the reaction mixture was treated
with 1 M NaOH (base hydrolysis) and the organic extract
was analyzedbyLC-MS. Production of the propargylated-
R-pyrone 9 was observed verifying that the KS domain can
indeed tolerate the bulkier propargyl substituent in the
diketide and extend it to the corresponding triketide
product (Figure 1).14 To confirm the structure of 9 synthe-
sized by CazF, a synthetic standard was prepared using a
4-step sequence (Scheme 2).15 Alkylation of commercially
available 2,4-pentanedione with 3-bromoprop-1-yn-1-yl-
trimethylsilane provided the dione 10, which in turn, was
elaborated to the β,δ-diketoester 11 using sodium bis-
(trimethylsilyl)amide and dimethyl carbonate. A base-
mediated cyclization of 11 with DBU yielded pyrone 12,
which underwent alkyne deprotection to furnish 9.
The higher amount of des-methylated compound 7
observed in the assay using 2 instead of 1 does suggest that
(14) In Figure 1 trace ii, there is an additional peak at 14.5 min. Based
on its m/z, this compound is most likely propargylic Se-adenosyl.
(15) For specific details on the synthetic reactions, see the Supporting
Information.
(16) Huisgen, R. 1,3-Dipolar Cycloaddition Chemistry; Padwa, A.,
Ed.; Wiley: New York, 1984.
Org. Lett., Vol. XX, No. XX, XXXX
C

Scheme 3. Biosynthesis of Chaetoviridin A (15) and 40-Propargyl-chaetoviridin A (17) Using the HR-PKS CazF, the Acyltransferase
CazE, and the caz Pyranoquinone Intermediate Cazisochromene (14) in the Presence of the Alkylating Cofactor 1 or 2
is identical to the expected mass of the triazole-containing
molecule 18 that forms as a result of alkyne-mediated
cycloaddition. Taken together, our experiments show that
the propargyl functionality installed by the MT domain
can indeed be propagated throughout the downstream
reaction steps, and be found in the final natural product
analogue.
In summary, we demonstrated that the auxiliary MT
domain within the highly reducing fungal PKS CazF can
act as a gateway for introducing orthogonally reactive
functional groups into polyketides. Using the promiscuous
nature of the MT domain, our study unveils an alternative
approach for enhancing the structural diversity of natural
products and opens the door for accessing and exploiting
MT domains in other natural product systems.
ꢁ
Acknowledgment. J.M.W. is supported by a L’Oreal
USA Fellowship for Women In Science, and G.C. is sup-
ported by a National Science Foundation Graduate Research
Fellowship (DGE-0707424). This work is supported by the
US National Institutes of Health (1DP1GM106413) to Y.T.
and (1DP2OD007335 and 1R01GM096056) to M.L. These
studies were supported by shared instrumentation grants
from the NSF (CHE-1048804) and the National Center for
Research Resources (S10RR025631).
Figure 2. Analysis of the enzymatically synthesized chaetovir-
idins. (A) HPLC analysis (360 nm) of chaetoviridins biosynthe-
sized in vitro when (i) CazF and CazE were incubated with 14,
malonyl-CoA, NADPH, and 1 and (ii) CazF and CazE were
incubated with 14, malonyl-CoA, NADPH, and 2. (B) HPLC
analysis (550 nM) of the triazole-containing 18 after labeling 17
with the rhodamine azide.
Supporting Information Available. Experimental proce-
dures, enzymatic reactions, detailed synthetic reactions, and
NMR spectroscopic data. This material is available free of
charge via the Internet at http://pubs.acs.org.
new peak 18 together with the disappearance of 17 was
detected by LC-MS (Figure 2B). This new product exhib-
ited UV absorption λmax at 550 nm, which is charcteristic
of the rhodamine dye; and a [M þ H]þ m/z = 1087, which
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX