Enzymatic formation of unnatural novel polyketides from alternate starter and nonphysiological extension substrate by chalcone synthase.

Article abstract of DOI:10.1021/ol0300165

[reaction: see text] In the chalcone synthase (CHS) enzyme reaction, both the starter molecule and the extension unit of the polyketide chain elongation reaction were simultaneously replaced with nonphysiological substrates. When incubated with benzoyl-CoA and methylmalonyl-CoA as substrates, recombinant CHS from Scutellaria baicalensis afforded an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide, 4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as substrates to produce an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.

Full text of DOI:10.1021/ol0300165

ORGANIC
LETTERS
2003
Vol. 5, No. 8
1277-1280
Enzymatic Formation of Unnatural Novel
Polyketides from Alternate Starter and
Nonphysiological Extension Substrate
by Chalcone Synthase
Ikuro Abe,* Yusuke Takahashi, Weiwei Lou, and Hiroshi Noguchi
UniVersity of Shizuoka, School of Pharmaceutical Sciences, and The 21st Century COE
Program, 52-1 Yada, Shizuoka 422-8526, Japan
abei@ys7.u-shizuoka-ken.ac.jp
Received February 4, 2003
ABSTRACT
In the chalcone synthase (CHS) enzyme reaction, both the starter molecule and the extension unit of the poyketide chain elongation reaction
were simultaneously replaced with nonphysiological substrates. When incubated with benzoyl-CoA and methylmalonyl-CoA as substrates,
recombinant CHS from Scutellaria baicalensis afforded an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with
a tetraketide, 4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the other hand, the enzyme also accepted hexanoyl-
CoA and methylmalonyl-CoA as substrates to produce an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.
Chalcone synthase (CHS) (EC 2.3.1.74) is a plant-specific
type III polyketide synthase (PKS) that catalyzes a stepwise
condensation of the C6-C3 unit of 4-coumaroyl-CoA (1a)
as a starter with three C2 units from malonyl-CoA (2)
(Scheme 1A).1,2 The reaction is initiated by binding of the
CoA-linked starter molecule to an active site cysteine. After
three rounds of malonyl-CoA decarboxylation and polyketide
chain extension reaction, regiospecific Claisen-type cycliza-
tion and aromatization of the enzyme-bound tetraketide inter-
mediate leads to formation of 4,2′,4′,6′-tetrahydroxychalcone
(naringenin chalcone) (5a) (Scheme 1A). In CHS enzyme
reactions in vitro, a triketide and a tetraketide pyrone, bis-
noryangonin (BNY) (3a)3 and 4-coumaroyltriacetic acid lac-
tone (CTAL) (4a)4 are also obtained as early-released derail-
ment byproducts. Recent crystallographic and structure-based
mutagenesis studies on alfalfa (Medicago satiVa) CHS2 and
other CHS-superfamily enzymes revealed the structural de-
tails of the active site governing the polyketide formation
reaction.5
In previous studies, we have demonstrated that CHS from
Scutellaria baicalensis has remarkably broad substrate
specificity toward either the starter molecule or the extension
unit of the polyketide chain elongation reaction.6,7 Thus,
(3) Kreuzaler, F.; Hahlbrock, K. Arch. Biochem. Biophys. 1975, 169,
84-90.
(4) Akiyama, T.; Shibuya, M.; Liu, H.-M.; Ebizuka, Y. Eur. J. Biochem.
1999, 263, 834-839.
(5) (a) Ferrer, J.-L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.; Noel, J.
P. Nature Struct. Biol. 1999, 6, 775-783. (b) Jez, J. M.; Ferrer, J.-L.;
Bowman, M. E.; Dixon, R. A.; Noel, J. P. Biochemistry 2000, 39, 890-
902. (c) Jez, J. M.; Noel, J. P. J. Biol. Chem. 2000, 275, 39640-39646. (d)
Jez, J. M.; Bowman, M. E.; Noel, J. P. Biochemistry 2001, 40, 14829-
14838. (e) Jez, J. M.; Bowman, M. E.; Noel, J. P. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 5319-5324. (f) Jez, J. M.; Austin, M. B.; Ferrer, J.;
Bowman, M. E.; Schro¨der, J.; Noel, J. P. Chem. Biol. 2000, 7, 919-930.
* To whom correspondence should be addressed. Phone/Fax: +81-54-
264-5662.
(1) Schro¨der, J. In ComprehensiVe Natural Products Chemistry; Sankawa,
U., Ed.; Elsevier: Oxford, 1999; Vol. 1, pp 749-771.
(2) Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 79-110.
10.1021/ol0300165 CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/22/2003

Scheme 1. Enzymatic Formation of Naringenin Chalcone (5a) from 4-Coumaroyl-CoA (1a) and Malonyl-CoA (2) and Formation of
Unnatural Polyketides from Alternate Starter (1b and 1c) and Nonphysiological Extension Substrates (6) by Recombinant S. baicalensis
CHS
instead of 4-coumaroyl-CoA, the enzyme accepted a wide
variety of starter molecules, both aromatic and aliphatic CoA
esters of different chain length, including benzoyl-CoA (1b)
and hexanoyl-CoA (1c), and efficiently performed sequential
decarboxylative condensations with malonyl-CoA (2) to
produce a series of chemically and structurally different
unnatural polyketides (Scheme 1B and 1C).6 On the other
hand, instead of malonyl-CoA, the enzyme also accepted
methylmalonyl-CoA (6) as an extension unit of the poyke-
tide chain elongation reaction. When incubated with 4-cou-
maroyl-CoA (1a) and methylmalonyl-CoA (racemic) (6),
S. baicalensis CHS afforded an unnatural C6-C5 aromatic
diketide (7a) along with a BNY- and a CTAL-type pyrone
byproduct, 8a and 9a (Scheme 1A).7 The extraordinary broad
substrate specificity of the plant PKS enabled us to develop
a chemical library of unnatural novel polyketides. In this
paper, in order to further examine the versatility of the
catalytic function of the enzyme, we carried out enzymatic
conversion experiments in which both the starter and the
extension unit of the CHS enzyme reaction were simulta-
neously replaced with nonphysiological substrates.
When recombinant S. baicalensis CHS8 was incubated
with benzoyl-CoA (1b) as a starter and methylmalonyl-CoA
(racemic) (6) as an extension substrate, two products were
isolated by reverse-phase HPLC (Figure 1A).9 As in the case
of previously reported enzyme reactions with methylmalonyl-
(7) Abe, I.; Takahashi, Y.; Noguchi, H. Org. Lett. 2002, 4, 3623-3626.
(8) Recombinant S. baicalensis CHS with an additional hexahistidine
tag at the C-terminal was expressed in E. coli and purified by Ni-chelate
affinity chromatography as described before. The KM and kcat values were
36.1 µM and 1.26 min-1, respectively, for 4-coumaroyl-CoA and 22.9 µM
and 0.83 min-1, respectively, for malonyl-CoA.
(9) The reaction mixture contained 54 µM of benzoyl-CoA (or hexanoyl-
CoA), 108 µM of methylmalonyl-CoA, and 20 µg of the purified
recombinant S. baicalensis CHS in a final volume of 500 µL of 100 mM
potassium phosphate buffer, pH 8.0, containing 1 mM EDTA. Incubations
were carried out at 30 °C for 18 h and stopped by addition of 50 µL of
20% HCl. The products were then extracted with 600 µL of ethyl acetate
and separated by reverse-phase HPLC (column, TSK-gel ODS-80Ts, 4.6
× 150 mm, Tosoh Co., Ltd., Japan; flow rate, 0.8 mL/min; UV detection
at 290 nm). The HPLC eluent for separation of 8b and 9b was 50% aqueous
MeOH containing 0.05% TFA, while for separation of 8c, gradient elution
was performed with H2O and MeOH containing 0.05% TFA (0-20 min,
linear gradient from 55 to 75% MeOH; 20-30 min, linear gradient from
75 to 80% MeOH). For large-scale enzyme reactions, benzoyl-CoA (15.0
mg, 17.3 µmol) and methylmalonyl-CoA (30.0 mg, 34.6 µmol) were
incubated with 52.6 mg of purified recombinant CHS in 300 mL of 100
mM phosphate buffer, pH 8.0, containing 1 mM EDTA at 30 °C for 18 h.
On the other hand, hexanoyl-CoA (5.0 mg, 5.8 µmol) and methylmalonyl-
CoA (10.0 mg, 11.5 µmol) were incubated with 20.0 mg of purified
recombinant CHS in 100 mL of the buffer at 30 °C for 18 h. The enzyme
still retained ca. 20% of activity after 18 h at 30 °C.
(6) (a) Abe, I.; Morita, H.; Nomura, A.; Noguchi, H. J. Am. Chem. Soc.
2000, 122, 11242-11243. (b) Morita, H.; Takahashi, Y.; Noguchi, H.; Abe,
I. Biochem. Biophys. Res. Commun. 2000, 279, 190-195. (c) Morita, H.;
Noguchi, H.; Schro¨der, J.; Abe, I. Eur. J. Biochem. 2001, 268, 3759-3766.
1278
Org. Lett., Vol. 5, No. 8, 2003

parent ion peak at m/z 273, indicating that the polyketide
chain elongation reaction had terminated after two or three
condensations with the C3 units from methylmalonyl-CoA.
Further confirmation of the structures was obtained by NMR
spectroscopic data of 8b and 9b isolated from a large-scale
enzyme incubation (0.5 mg each, 7 and 6% yields from 15.0
mg of benzoyl-CoA, respectively).9 The 1H NMR spectrum
of 8b showed two methyl singlets (δ 2.04 and 1.96) of a
R-pyrone ring, while that of 9b revealed two methyl singlets
(δ 2.05 and 1.84) and one methyl doublet (δ 1.46, J ) 6.7
Hz) coupled with a proton (δ 4.85, J ) 6.7 Hz), in addition
to the benzene ring proton signals. Moreover, the homo-
nuclear (1H-1H COSY) and heteronuclear (HMQC and
HMBC) correlation spectra were uniquely consistent with
the proposed structures of the unnatural novel methylated
triketide 8b and tetraketide 9b.
On the other hand, when incubated with hexanoyl-CoA
(1c) and methylmalonyl-CoA as substrates, S. baicalensis
CHS afforded 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one
(8c)12 as a single product (Figure 1B).9 The LC-ESIMS
spectrum of 8c gave a parent ion peak [M + H]+ at m/z
211, indicating the polyketide chain elongation reaction had
terminated after two condensations). The 1H NMR spectrum
of 8c obtained from a large-scale enzyme reaction (0.5 mg
each, 22% yield from 5.0 mg of hexanoyl-CoA)9 showed
two methyl singlets (δ 1.93 and 1.89) of an R-pyrone ring
in addition to the aliphatic chain proton signals. Furthermore,
the homonuclear (1H-1H COSY) and heteronuclear (HMQC
and HMBC) correlation spectra conclusively supported the
structure.
This is the first demonstration of the enzymatic formation
of unnatural polyketides from alternate starter and nonphysi-
ological extension substrates by a plant PKS. The enzyme
accepted benzoyl-CoA and methylmalonyl-CoA as substrates
and catalyzed the formation of a novel methylated triketide
8b as well as a tetraketide pyrone 9b13 (Scheme 1B), while
hexanoyl-CoA and methylmalonyl-CoA afforded a novel
(10) HPLC: Rt ) 22.7 min. LC-ESIMS: m/z 217 [M + H]+. UV: λmax
232 and 310 nm. 1H NMR (500 MHz, CD3OD): δ 7.87 (2H, ddd, J ) 8.2
Hz, 1.2 Hz, 1.2 Hz, H-2′ and H-6′), 7.46 (3H, m, H-3′, H-4′, and H-5′),
2.04 (3H, s, Me-5), 1.96 (3H, s, Me-3). 13C NMR (125 MHz, CD3OD): δ
170.2 (C-4), 168.6 (C-2), 156.0 (C-6), 134.5 (C-1′), 129.3 (C-4′), 128.5
(C-2′ and C-6′), 128.1 (C-3′ and C-5′), 111.4 (C-5), 98.2 (C-3), 10.6
(Me-5), 7.8 (Me-3). HRMS (FAB): found for [C13H12O3 + Na]+, 239.0669;
calcd, 239.0684.
Figure 1. HPLC profile of the enzyme reaction products (A) from
benzoyl-CoA (1b) and methylmalonyl-CoA (6) and (B) from
hexanoyl-CoA (1c) and methylmalonyl-CoA (6). 4-Hydroxy-3,5-
dimethyl-6-phenyl-pyran-2-one (8b) (Rt ) 22.7 min), 4-hydroxy-
3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one (9b)
(Rt ) 12.9 min), and 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one
(8c) (Rt ) 20.8 min).
(11) HPLC: Rt ) 12.9 min. LC-ESIMS: m/z 273 [M + H]+. UV: λmax
246 and 290 nm. 1H NMR (500 MHz, CD3OD): δ 7.87 (2H, ddd, J ) 7.0
Hz, 1.0 Hz, 1.0 Hz, H-2′′ and H-6′′), 7.56 (1H, ddd, J ) 7.4 Hz, 7.4 Hz,
1.0 Hz, H-4′′), 7.45 (2H, ddd, J ) 7.4 Hz, 7.0 Hz, 1.0 Hz, H-3′′ and H-5′′),
4.85 (1H, t, J ) 6.7 Hz, H-1′), 2.05 (3H, s, Me-5), 1.84 (3H, s, Me-3), 1.46
(3H, d, J ) 6.7 Hz, Me-1′). 13C NMR (125 MHz, CD3OD): δ 198.4 (C-
2′), 167.4 (C-4), 167.4 (C-2), 157.6 (C-6), 137.0 (C-1′′), 133.0 (C-4′′), 128.6
(C-3′′ and C-5′′), 127.9 (C-2′′ and C-6′′), 109.8 (C-5), 98.0 (C-3), 43.8
(C-1′), 13.4 (Me-1′), 8.5 (Me-5), 7.5 (Me-3). HRMS (FAB): found for
[C16H16O4 + Na]+, 295.0956; calcd, 295.0946.
CoA as a substrate, formation of the nonphysiological
products was so slow (apparently at least 100 times slower
than that of chalcone) that we carried out the incubation for
18 h.7,9
The two products showed UV spectra similar to those of
BNY and CTAL, respectively, suggesting the structures of
4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one (8b)10 and
4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-
pyran-2-one (9b),11 respectively (Scheme 1B). The LC-
ESIMS spectrum of BNY-type 8b gave a parent ion peak
[M + H]+ at m/z 217, while that of CTAL-type 9b gave a
(12) HPLC: Rt ) 20.8 min. LC-ESIMS: m/z 211 [M + H]+. UV: λmax
1
290 nm. H NMR (500 MHz, CD3OD): δ 2.53 (t, 1H, J ) 7.4 Hz, H-1′),
1.93 (s, 3H, Me-5), 1.89 (s, 3H, Me-3), 1.64 (m, 2H, H-2′), 1.34 (m, 4H,
H-3′ and H-4′), 0.90 (t, 3H, J ) 6.9 Hz, H-5′). 13C NMR (125 MHz, CD3-
OD): δ 172.9 (C-2), 167.8 (C-4), 159.4 (C-6), 109.3 (C-5), 98.6 (C-3),
31.0 (C-3′), 30.0 (C-1′), 26.5 (C-2′), 21.7 (C-4′), 12.7 (C-5′), 8.7 (Me-5),
7.5 (Me-3). HRMS (FAB): found for [C12H18O3 + Na]+, 233.1148; calcd,
233.1153.
(13) Tetraketide pyrone 9b was obtained as a racemic compound.
Utilization of chiral methylmalonyl-CoA as a substrate would provide useful
information on the stereochemistry of the enzyme reaction.
Org. Lett., Vol. 5, No. 8, 2003
1279

methylated triketide pyrone 8c (Scheme 1C).14 The structures
of the reaction products were apparently different from that
of chalcone, the regular product of CHS. The extraordinary
broad substrate specificity and the versatility of catalytic
function of the recombinant S. baicalensis CHS were quite
remarkable. The in vitro promiscuity of the enzyme seems
to be a common feature of type III PKS enzymes of plant
and bacterial origin.1,2 Recently, it has been reported that
RppA, a bacterial type III PKS that normally catalyzes
formation of 1,3,6,8-tetrahydroxynaphthalene from five
molecules of malonyl-CoA, also showed broad substrate
specificity and accepted methylmalonyl-CoA and acetoacetyl-
CoA to produce a C-methylated pyrone, 3,6-dimethyl-4-
hydroxy-2-pyrone.15 Furthermore, CHS2 from Pinus strobes
has been reported to catalyze a single extension of a diketide
intermediate mimic to a triketide pyrone using methylma-
lonyl-CoA.16
product (Scheme 1A).7 Presumably, loading of the less bulky
starter molecule of benzoyl-CoA or hexanoyl-CoA into the
coumaroyl-binding pocket of the active site enabled further
polyketide chain elongation reactions (Scheme 1B). In the
case of the aliphatic hexanoyl-CoA, the chain extension was
interrupted at the triketide stage and afforded the triketide
pyrone as a single product. As mentioned above, due to the
presence of the additional bulky methyl group of the
extension unit analogue, the enzyme reactions with meth-
ylmalonyl-CoA were extremely slow compared with those
with malonyl-CoA, the normal extension substrate. For
further production of unnatural novel poyketides, manipula-
tion of the plant PKS reactions by utilizing substrate
analogues and rationally engineered mutant enzymes is now
in progress in our laboratories.
Acknowledgment. We thank Drs. Hiroaki Utsumi and
Takako Fujimoto (JEOL) for NMR measurements. This work
was in part supported by the 21st Century COE Program
and Grant-in-Aid for Scientific Research (Nos. 14580613
and 13480188) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan, and by a Grant-in-
Aid from NOVARTIS Foundation (Japan) for the Promotion
of Science and from the Tokyo Biochemical Research
Foundation.
Interestingly, formation of neither diketides nor tetraketides
with a new aromatic ring system was detected in the assay
mixtures. In contrast, as we have reported in a previous paper,
when 4-coumaroyl-CoA (1a), the normal starter substrate,
was incubated with methylmalonyl-CoA, most of the
polyketide chain elongation reactions were terminated at the
diketide stage and afforded phenylpentanone (7a) as a major
(14) Similar product pattern was observed for recombinant stilbene
synthase (STS) from Arachis hypogaea, another plant type III PKS that
normally catalyzes formation of resveratrol from 4-coumaroyl-CoA and
malonyl-CoA. Furthermore, our preliminary results showed that both S.
baicalensis CHS and A. hypogaea STS accepted cinnamoyl-CoA, pheny-
lacetyl-CoA, and isovaleryl-CoA as a starter substrate to produce methylated
BNY- and CTAL-type pyrones.
(15) Funa, N.; Ohnishi, Y.; Ebizuka, Y.; Horinouchi, S. J. Biol. Chem.
2002, 277, 4628-4635.
(16) Schro¨der, J.; Raiber, S.; Berger, T.; Schmidt, A.; Schmidt, J.; Soares-
Sello, A. M.; Bardshiri, E.; Strack, D.; Simpson, T. J.; Veit, M.; Schro¨der,
G. Biochemistry 1998, 37, 8417-8425.
Supporting Information Available: Set of NMR spectra
1
(1H NMR, H-1H COSY, HMQC, and HMBC) of 4-hy-
droxy-3,5-dimethyl-6-phenyl-pyran-2-one (8b), 4-hydroxy-
3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-
one (9b), and 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one
(8c). This material is available free of charge via the Internet
at http://pubs.acs.org.
OL0300165
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