Enzymatic formation of an unnatural C6-C5 aromatic polyketide by plant type III polyketide synthases

Article abstract of DOI:10.1021/ol0201409

(graph presented) Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural C₆-C₅ aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In contrast, succinyl-CoA was not accepted as a substrate by the enzymes.

Full text of DOI:10.1021/ol0201409

ORGANIC
LETTERS
2002
Vol. 4, No. 21
3623-3626
Enzymatic Formation of an Unnatural
C −C Aromatic Polyketide by Plant
6
5
Type III Polyketide Synthases
Ikuro Abe,* Yusuke Takahashi, and Hiroshi Noguchi
UniVersity of Shizuoka, School of Pharmaceutical Sciences, 52-1 Yada,
Shizuoka 422-8526, Japan
abei@ys7.u-shizuoka-ken.ac.jp
Received July 22, 2002
ABSTRACT
Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the
polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from
Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural
C −C aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In
6
5
contrast, succinyl-CoA was not accepted as a substrate by the enzymes.
Type III poyketide synthases (PKSs) are pivotal enzymes
for construction of the basic skeletons of plant polyketides
with diverse structures and biological activities. Chalcone
synthase (CHS) (EC 2.3.1.74) and stilbene synthase (STS)
(EC 2.3.1.95) catalyze a sequential condensation of the C6-
C3 unit of 4-coumaroyl-CoA (1) as a starter with three C2
units from malonyl-CoA (2) (Scheme 1).1 After three rounds
of the polyketide chain elongation reaction, cyclization and
aromatization of the enzyme-bound tetraketide intermediate
leads to formation of 4,2′,4′,6′-tetrahydroxychalcone (nar-
ingenin chalcone) (3) and trans-3,4′,5-trihydroxystilbene
(resveratrol) (4), respectively. Further, in enzyme reactions
in vitro, pyrone derivatives 4-coumaroyltriacetic acid lactone
(CTAL) (5)2 and bis-noryangonin (BNY) (6)3 are formed as
early-released derailment byproducts. On the other hand,
recently reported benzalacetone synthase (BAS) (EC 2.3.1.-)
catalyzes a one-step decarboxylative condensation of 4-cou-
maroyl-CoA with malonyl-CoA to produce the C6-C4
skeleton of benzalacetone (7) (Scheme 1), a precursor of
medicinally important phenylbutanoids such as antiinflam-
matory lindleyin and gingerols.4
The plant PKSs are homodimeric enzymes sharing 60-
75% amino acid sequence identity with other members of
the CHS-superfamily enzymes, including 2-pyrone synthase
(2PS)5 and acridone synthase (ACS).6 Recent crystallographic
and structure-based mutagenesis studies on Medicago satiVa
CHS27 and Gerbera hybrida 2PS8 revealed the structural
details of the active site govering the enzyme reaction, i.e.,
starter molecule loading, malonyl-CoA decarboxylation,
polyketide chain elongation, and regiospecificities of the
(4) Abe, I.; Takahashi, Y.; Morita, H.; Noguchi, H. Eur. J. Biochem.
2001, 268, 3354-3359.
(5) Eckermann, S.; Schro¨der, G.; Schmidt, J.; Strack, D.; Edrada, R. A.;
Helariutta, Y.; Elomaa, P.; Kotilainen, M.; Kilpela¨inen, I.; Proksch, P.; Teeri,
T. H.; Schro¨der, J. Nature 1998, 396, 387-390.
(6) (a) Lukacin, R.; Springob, K.; Urbanke, C.; Ernwein, C.; Schro¨der,
G.; Schro¨der, J.; Matern, U. FEBS Lett. 1999, 448, 135-140. (b) Springob,
K.; Lukacin, R.; Ernwein, C.; Groning, I.; Matern, U. Eur. J. Biochem.
2000, 267, 6552-6559.
(7) (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.
* To whom correspondence should be addressed.
(1) Schro¨der, J. In ComprehensiVe Natural Products Chemistry: Sankawa,
U., Ed.; Elsevier: Oxford, 1999; Vol. 1, pp 749-771.
(2) Akiyama, T.; Shibuya, M.; Liu, H.-M.; Ebizuka, Y. Eur. J. Biochem.
1999, 263, 834-839.
(3) Kreuzaler, F.; Hahlbrock, K. Arch. Biochem. Biophys. 1975, 169,
84-90.
(8) Jez, J. M.; Austin, M. B.; Ferrer, J.; Bowman, M. E.; Schro¨der, J.;
Noel, J. P. Chem. Biol. 2000, 7, 919-930.
10.1021/ol0201409 CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/13/2002

Scheme 1. Proposed Mechanism for the conversion of 4-Coumaroyl-CoA (1) and Malonyl-CoA (2) to Naringenin Chalcone (3) by
CHS, to Resveratrol (4) by STS, and Benzalacetone (5) by BAS.a
a Pyrone derivatives, BNY (6), and CTAL (7) are common byproducts of the CHS and STS enzyme reactions in vitro.
cyclization reactions. The catalytic center of the M. satiVa
CHS2 enzyme is composed of four amino acid residues
(Cys164, Phe215, His303, and Asn336) well conserved in
all the known type III PKSs. The only exception is the BAS
that lacks the active site Phe215 and has a Leu at this
position.4 Phe215 has been proposed to facilitate malonyl-
CoA decarboxylation and help orient substrates and inter-
mediates during the sequential condensation reactions in
CHS.7b,e These hypotheses may explain why the polyketide
chain elongation reaction is terminated at the diketide stage
in BAS.
structurally different unnatural polyketides. Manipulation of
the enzyme reaction through the use of artificial substrates
thus led to development of a chemical library of unnatural
novel polyketides.
In addition to the starter substrate specificity, it is also
interesting to test if the enzymes can accept analogues of
the extension unit of the sequential condensation reactions.
We now describe enzymatic reactions utilizing methyl-
malonyl-CoA (8) and succinyl-CoA (9), both having one
more carbon atom than malonyl-CoA (2), by plant PKS
enzymes. We also reasoned that the nucleophilicity of the
R-carbon atom of succinyl-CoA (9) is significantly lower
compared with that of malonyl-CoA having a â-carbonyl
group, and succinyl-CoA may be accepted by the enzymes
and activated for the decarboxylative Claisen condensation
reactions. Evidence favoring selection of methylmalonyl-CoA
as a starter has been obtained for CHS2 from Pinus strobes.11
This enzyme was reported to be completely inactive with
malonyl-CoA as a substrate; however, it catalyzed a one-
step condensation of methylmalonyl-CoA (8) with a diketide
derivative (N-acetylcysteaminethioester of 3-oxo-5-phenyl-
In previous studies, we have demonstrated that CHSs from
Scutellaria baicalensis9 and STS from Arachis hypogaea10
have remarkably broad substrate specificity toward the starter
molecule of the enzyme reaction. Instead of 4-coumaroyl-
CoA, the enzymes accepted disparate starter molecules, both
aromatic and aliphatic CoA esters of different chain length,
and efficiently performed sequential condensation and cy-
clization reactions to produce a series of chemically and
(9) (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.
(10) Morita, H.; Noguchi, H.; Schro¨der, J.; Abe, I. Eur. J. Biochem. 2001,
268, 3759-3766.
(11) 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.
3624
Org. Lett., Vol. 4, No. 21, 2002

pent-4-enoic acid) to produce a methylated triketide styrylpy-
rone. Here in this letter, we now report on the detailed
enzymatic conversion studies of the extension unit analogues
by (i) CHS from S. baicalensis, (ii) STS from A. hypogaea,
and (iii) BAS from Rheum palmatum.12
When incubated with methylmalonyl-CoA (racemic) (8)
and 4-coumaroyl-CoA (1) as substrates, both S. baicalensis
CHS and A. hypogaea STS afforded 1-(4-hydroxyphenyl)-
pent-1-en-3-one (11) as a major product along with the BNY-
type (12) and the CTAL-type (13) pyrone byproducts, while
R. palmatum BAS yielded the C6-C5 aromatic polyketide
(11) as a single product (Figure 1).13 Here 4-coumaric acid
(10) was also obtained as a result of hydrolysis of 1. It should
be noted that formation of the products were detected only
after 12 h of incubation, instead of 20 min of incubation for
the standard assay with malonyl-CoA. The formation of the
phenylpentenone was thus apparently at least 100 times
slower than that of regular products of the enzymes (CHS,
STS, and BAS).13
Confirmation of the structure of the major product 11 was
obtained as follows.14 First, the LC-ESIMS spectrum of the
product gave a parent ion peak [M + H]+ at m/z 177, and
its UV spectrum with a λmax at 322 nm was similar to that
of benzalacetone (7). Further, the 1H NMR spectrum of the
product obtained from a large-scale enzyme reaction (69%
yield from 7.5 mg of 4-coumaroyl-CoA) showed A2B2-type
(δ 7.40, 2H, d, J ) 8.4 Hz, and 6.71, 2H, d, J ) 8.4 Hz)
aromatic signals together with a pair of trans-coupled R,â-
unsaturated olefinic protons (δ 7.48 and 6.56, each 1H, d, J
Figure 1. HPLC profile of the enzyme reaction products from
4-coumaroyl-CoA (1) and methylmalonyl-CoA (racemic) (8) by
(A) S. baicalensis CHS, (B) A. hypogaea STS, and (C) R. palmatum
BAS. 4-Coumaric acid (10) (Rt ) 3.7 min), 1-(4-hydroxyphenyl)-
pent-1-en-3-one (11) (Rt ) 9.8 min), the BNY-type pyrone (12)
(Rt ) 20.0 min), and the CTAL-type pyrone (13) (Rt ) 11.6 min).
Here 4-coumaric acid (10) was obtained as a result of hydrolysis
of 1.
(12) Recombinant S. baicalensis CHS,9 A. hypogaea STS,10 and R.
palmatum BAS4 with an additional hexahistidine tag at the C-terminal were
expressed in E. coli and purified by Ni-chelate affinity chromatography as
described before. The purified enzymes showed the following KM and kcat
values for 4-coumaroyl-CoA: CHS (36.1 µM and 1.26 min-1), STS (11.2
µM and 1.20 min-1), and BAS (10.0 µM and 1.79 min-1).
) 16.4 Hz) and R-ethyl protons (2.61, 2H, q, J ) 7.2 Hz,
and 1.02, 3H, t, J ) 7.2 Hz). The structure was finally
verified by HMQC and HMBC experiments. On the other
hand, the structures of the two byproducts were respectively
determined to be the BNY-type (12)15 and the CTAL-type
pyrone (13)16 on the basis of the LC-ESIMS and UV spectra.
The LC-ESIMS spectrum of 12 gave a parent ion peak [M
+ H]+ at m/z 259, and in MS/MS (precursor ion at m/z 259)
the fragment at m/z 215 corresponded to [M + H - CO2]+,
while that of 13 gave a parent ion peak [M + H]+ at m/z
315, and in MS/MS (precursor ion at m/z 315) the fragment
(13) The reaction mixture contained 54 µM 4-coumaroyl-CoA, 108 µM
methylmalonyl-CoA (racemic), and 20 µg of the purified recombinant
enzyme 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;
eluent, 50% aqueous MeOH containing 0.05% TFA; flow rate, 0.8 mL/
min). For large-scale enzyme reactions, 4-coumaroyl-CoA (7.5 mg, 8.2
µmol) and methylmalonyl-CoA (racemic, 15.0 mg, 17.3 µmol) were
incubated with 50 mg of purified recombinant CHS in 750 mL of 100 mM
phosphate buffer, pH 8.0, containing 1 mM EDTA at 30 °C for 18 h. The
apparent KM and kcat values for methylmalonyl-CoA for the formation of
the unnatural C6-C5 aromatic polyketide (11) are as follows: CHS (129
µM and 0.0103 min-1), STS (79.3 µM and 0.0194 min-1), and BAS (50.4
µM and 0.0360 min-1).
(14) HPLC: Rt ) 9.8 min. LC-ESIMS: m/z 177 [M + H]+. UV: λmax
322 nm. 1H NMR (400 MHz, CD3OD): δ 7.48 (H-1, d, J ) 16.4 Hz), 7.40
(H-2′ and H-6′, d, J ) 8.4 Hz), 6.71 (H-3′ and H-5′, d, J ) 8.4 Hz), 6.56
(H-2, d, J ) 16.4 Hz), 2.61 (CH2, q, J ) 7.2 Hz), 1.02 (CH3, t, J ) 7.2
Hz). 13C NMR (100 MHz, CD3OD): δ 203.9 (C-3), 161.6 (C-1), 144.8
(C-4′), 131.4 (C-2′ and C-6′), 127.1 (C-2), 123.6 (C-1′), 116.9 (C-3′ and
C-5′), 34.3 (C-4), 8.8 (C-5). HRMS (FAB): found for [C11H13O2]+
177.0919, calcd 177.0916.
(15) HPLC: Rt ) 20.0 min. LC-ESIMS: m/z 259 [M + H]+, MS/MS
(precursor ion at m/z 259), m/z 229 (100) [M + H - 2Me]+, 215 (51) [M
+ H - CO2]+, 140 (23), 119 (27). UV: λmax 370 nm. HRMS (FAB): found
for [C15H15O4]+ 259.0981, calcd 259.0970.
(16) HPLC: Rt ) 11.6 min. LC-ESIMS: m/z 315 [M + H]+, MS/MS
(precursor ion at m/z 315), m/z 271 (13) [M + H - CO2]+, 195 (11), 147
(100), 119 (37). UV: λmax 332 nm. HRMS (FAB): found for [C18H19O5]+
315.1220, calcd 315.1232.
Org. Lett., Vol. 4, No. 21, 2002
3625

Scheme 2. Enzymatic Conversion of 4-Coumaroyl-CoA (1) and Methylmalonyl-CoA (8) to 1-(4-Hydroxyphenyl)pent-1-en-3-one
(11), the BNY-Type Pyrone (12), and the CTAL-Type Pyrone (13).
at m/z 271 corresponded to [M + H - CO2]+, confirming
the presence of R-pyrone ring. Furhermore, UV spectrum
of 12 (λmax at 370 nm) and 13 (λmax at 332 nm) respectively
showed good similarity to that of BNY (6) and CTAL (5),
supporting the structures.
than malonyl-CoA, the nucleophilicity of the R-carbon of
succinyl-CoA may not be high enough to perform the
decarboxylative Claisen condensation reaction.
In summary, this is the first demonstration of the enzymatic
formation of the unnatural C6-C5 aromatic polyketide by
plant PKSs. The enzymes accepted the nonphysiological
extension unit analogue in addition to a wide variety of starter
molecule analogues. Manipulation of the plant PKS enzyme
reactions by substrate analogues along with rationally
engineered mutant enzymes would lead to further production
of chemically and structurally disparate unnatural novel
polyketides, which is now in progress in our laboratories.
These experiments were the first to demonstrate that plant
PKSs can catalyze the formation of the unnatural C6-C5
aromatic polyketide (11) from methylmalonyl-CoA and
4-coumaroyl-CoA (Scheme 2). The C6-C5 skeleton was
produced by one-step decarboxylative condensation of the
C6-C3 unit of 4-coumaroyl-CoA with the C2 unit of
methylmalonyl-CoA, as in the case of the formation of the
C6-C4 benzalacetone (7) from malonyl-CoA and 4-couma-
royl-CoA. Thus, most of the polyketide chain elongation
reactions were terminated at the diketide stage presumably
due to the presence of the additional bulky methyl group of
the extension unit analogue. Moreover, it is noteworthy that
since diketide carboxylic acid was not detected in the reaction
mixture, decarboxylation of the enzyme-bound diketide
intermediate should be tightly controlled by the enzyme.
In contrast, when incubated with succinyl-CoA (9) and
4-coumaroyl-CoA (1) as substrates, none of the enzymes
(CHS, STS, and BAS) afforded any reaction products,
indicating succinyl-CoA was not accepted as a substrate for
the polyketide chain elongation reaction. In addition to the
steric factor that succinyl-CoA has one more carbon atom
Acknowledgment. The authors are indebted to Professor
Joachim Schro¨der (Universita¨t Freiburg) for the A. hypogaea
STS clone. This work was supported in part by a Grant-in-
Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
Supporting Information Available: Comparative NMR
data for 1-(4-hydroxyphenyl)pent-1-en-3-one (11) and ben-
zalacetone (7), NMR spectra of 11 (1H and 13C NMR,
HMQC, and HMBC), and LC/MS/MS data of the pyrone
byproducts 12 and 13. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL0201409
3626
Org. Lett., Vol. 4, No. 21, 2002