Enzymatic formation of long-chain polyketide pyrones by plant type III polyketide synthases

Article abstract of DOI:10.1016/j.phytochem.2004.08.005

Recombinant chalcone synthase from Scutellaria baicalensis and stilbene synthase from Arachis hypogaea accepted CoA esters of long-chain fatty acid as a starter substrate, and carried out sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide α-pyrones. Recombinant chalcone synthase (CHS) from Scutellaria baicalensis and stilbene synthase (STS) from Arachis hypogaea accepted CoA esters of long-chain fatty acid (CHS up to the C₁₂ ester, while STS up to the C₁₄ ester) as a starter substrate, and carried out sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide α-pyrones. Interestingly, the C₆, C₈, and C₁₀ esters were kinetically favored by the enzymes over the physiological starter substrate; the k _cat/K_M values were 1.2- to 1.9-fold higher than that of p-coumaroyl-CoA. The catalytic diversities of the enzymes provided further mechanistic insights into the type III PKS reactions, and suggested involvement of the CHS-superfamily enzymes in the biosynthesis of long-chain alkyl polyphenols such as urushiol and ginkgolic acid in plants.

Full text of DOI:10.1016/j.phytochem.2004.08.005

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2447–2453
www.elsevier.com/locate/phytochem
Enzymatic formation of long-chain polyketide pyrones by plant type
III polyketide synthases
*
Ikuro Abe , Tatsuya Watanabe, Hiroshi Noguchi
School of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received 23 April 2004; received in revised form 24 June 2004
Abstract
Recombinant chalcone synthase (CHS) from Scutellaria baicalensis and stilbene synthase (STS) from Arachis hypogaea accepted
CoA esters of long-chain fatty acid (CHS up to the C12 ester, while STS up to the C14 ester) as a starter substrate, and carried out
sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide a-pyrones. Interestingly, the C6, C8,
and C10 esters were kinetically favored by the enzymes over the physiological starter substrate; the kcat/KM values were 1.2- to 1.9-
fold higher than that of p-coumaroyl-CoA. The catalytic diversities of the enzymes provided further mechanistic insights into the
type III PKS reactions, and suggested involvement of the CHS-superfamily enzymes in the biosynthesis of long-chain alkyl poly-
phenols such as urushiol and ginkgolic acid in plants.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Chalcone synthase; Stilbene synthase; Type III polyketide synthase; Long-chain a-pyrones; Urushiol; Ginkgolic acid
1. Introduction
chain elongation reaction, cyclization of the enzyme-
bound tetraketide intermediate lead to formation of
4,20,40,60-tetrahydroxychalcone (naringenin chalcone)
(3) or trans-3,40,5-trihydroxystilbene (resveratrol) (4),
respectively (Scheme 1). In addition, in enzyme reactions
in vitro, a triketide and a tetraketide a-pyrone; bisnor-
yangonin (BNY) (5) (Kreuzaler and Hahlbrock,
1975a,b) and p-coumaroyltriacetic acid lactone (CTAL)
(6) (Akiyama et al., 1999), are also obtained as early re-
leased derailment by-products. Crystallographic and
site-directed mutagenesis studies on alfalfa (Medicago
sativa) CHS revealed the active site machinery of the
chalcone forming reaction which proceeds through star-
ter molecule loading at Cys164, malonyl-CoA decarb-
oxylation, polyketide chain elongation, followed by
cyclization and aromatization of the enzyme-bound tet-
raketide intermediate (Ferrer et al., 1999; Jez and Noel,
2000; Jez et al., 2000a,b, 2001, 2002; Tropf et al., 1995;
Suh et al., 2000a,b; Abe et al., 2001, 2003a,b). The
active-site of the enzyme is composed of the
The chalcone synthase (CHS) superfamily of type III
polyketide synthases (PKSs) are pivotal enzymes in the
biosynthesis of flavonoids as well as a wide range of
structurally diverse, biologically important natural
products (Schro¨der, 1999; Austin and Noel, 2003).
The homodimer of relatively modest-sized proteins of
40–45 kDa, sharing 25–90% amino acid sequence iden-
tity with each other, catalyze the assembly of complex
natural products by successive decarboxylative conden-
sations of malonyl-CoA in a biosynthetic process that
closely parallels fatty acid biosynthesis. CHS (EC
2.3.1.74) and stilbene synthase (STS) (EC 2.3.1.95) thus
perform sequential condensation of the C6–C3 unit of p-
coumaroyl-CoA (1) as a starter with three C2 units from
malonyl-CoA (2). After three rounds of the polyketide
*
Corresponding author. Tel./fax: +81 54 264 5662.
E-mail address: abei@ys7.u-shizuoka-ken.ac.jp (I. Abe).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.08.005

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I. Abe et al. / Phytochemistry 65 (2004) 2447–2453
Scheme 1. Proposed mechanism for the conversion of p-coumaroyl-CoA (1) and malonyl-CoA (2) to naringenin chalcone (3) and resveratrol (4) by
CHS and STS. The pyrone derivatives, bisnoryangonin (BNY) (5) and 4-coumaroyltriacetic acid lactone (CTAL) (6) are common by-products of the
enzyme reactions in vitro.
coumaroyl-binding pocket and the cyclization pocket,
defined by four residues conserved in the type III PKS
enzymes (Cys164, Phe215, His303, and Asn336).
The functional diversity and the promiscuity of the
CHS-superfamily enzymes are remarkable. In previous
studies, we demonstrated that recombinant CHS from
Scutellaria baicalensis (Labiatae) and STS from Arachis
hypogaea (Fabaceae) have unusually broad substrate
specificities toward the starter and the extender sub-
strate (Abe et al., 2000, 2001, 2002, 2003a,b; Morita
et al., 2000, 2001). Thus, instead of p-coumaroyl-CoA,
the enzymes accepted a variety of aromatic and aliphatic
CoA esters as a starter substrate, and efficiently yielded
a series of chemically and structurally different unnatu-
ral polyketides. Furthermore, both CHS and STS also
accepted methylmalonyl-CoA as an extension substrate
to catalyze formation of an unnatural C6–C5 aromatic
polyketide. The enzymes even afforded unnatural poly-
ketides when both the starter and the extender substrate
were simultaneously replaced with non-physiological
analogues.
and ginkgo tree (Ginkgo biloba, Ginkgoaceae), respec-
tively (Scheme 2) (Dewick, 2002). Further, it was re-
cently reported that a mycobacterial type III PKS
It has been postulated that CoA esters of long-chain
fatty acid such as palmitoleoyl (C16)–CoA can act as a
starter substrate for malonyl-CoA chain extension, lead-
ing to formation of alkyl polyphenols including urushiol
and ginkgolic acid (anacardic acid), the allergic sub-
stances of lacquer tree (Rhus verniciflua, Anacardiaceae)
Scheme 2. Proposed mechanism for the biosynthesis of urushiol and
ginkgolic acid (anacardic acid) from palmitoleoyl-CoA.

I. Abe et al. / Phytochemistry 65 (2004) 2447–2453
2449
from Mycobacterium tuberculosis, sharing 25–45% ami-
no acid sequence similarity with plant CHSs, catalyzed
formation of triketide and tetraketide a-pyrones from
long-chain fatty acyl-CoA (C12–C20) (Saxena et al.,
2003). These suggest possible involvement of the type
III CHS-superfamily enzymes also in the biosynthesis
of the alkyl polyphenols in plants. Considering the
broad substrate specificities of CHS and STS, it was thus
interesting to test whether the enzymes also accept the
CoA-esters of long-chain fatty acid as a starter of the
polyketide formation reactions. Here in this paper, we
describe enzymatic conversion of long-chain fatty acyl-
CoAs (C6–C18) by recombinant S. baicalensis CHS
and A. hypogaea STS. Interestingly, the C6, C8, and
C10 esters were kinetically favored by the type III
CHS-superfamily enzymes over the physiological starter
p-coumaroyl-CoA.
nol (1,3,5-trihydroxybenzene) (11) derivatives with a
newly formed aromatic ring system (Scheme 3) were
not detected in the reaction mixtures.
The structure of the enzyme reaction products were
unambiguously determined by UV and LC-ESIMS,
using 4-hydroxy-6-pentyl-2-pyrone (8a) and 4-hydroxy-
6-(2-oxoheptyl)-2-pyrone (9a) obtained in our previous
works, as authentic compounds (Morita et al., 2000,
2001). Each enzyme reaction product showed a UV
spectrum similar to either that of 8a or 9a. Further,
the LC-ESIMS spectrum gave a parent ion peak
[M ꢀ H]ꢀ indicating the polyketide formation reaction
had terminated after two (triketide lactone) or three (tet-
raketide lactone) condensations of malonyl-CoA. More-
over, in MS/MS, the fragment corresponding to
[M ꢀ H–CO2]ꢀ confirmed the presence of a a-pyrone
ring structure. The enzyme reactions were therefore ter-
minated without aromatic ring formation. Either an acid
catalyzed lactonization of linear derailment products in
solution, or the acid stabilization of lactones originally
formed within the active site of the enzyme, resulted in
the formation of the triketide and tetraketide a-pyrones.
In earlier work, it has been reported that the parsley
(Petroselinum horstense) CHS accepted butyryl (C4)-
CoA and hexanoyl (C6)-CoA as starter substrates in
pH dependent manner; formation of phloroglucinol
derivatives from these substrates was only observed at
2. Results and discussion
The TLC based assay as well as the LC-ESIMS anal-
yses of the enzyme reaction products revealed that both
recombinant S. baicalensis CHS and A. hypogaea STS
readily accepted the CoA esters of long-chain fatty acid
(7a–7e) as a starter substrate for the polyketide chain
elongation reactions (Fig. 1). S. baicalensis CHS ac-
cepted the C6–C12 ester, while A. hypogaea STS up to
the C14 ester, and carried out sequential decarboxylative
condensations with malonyl-CoA to yield triketide a-
pyrones (8a–8e) as major products. In this regard, the
STS showed broader substrate tolerance toward the
unnatural substrates than the CHS. Formations of tet-
raketide a-pyrones (9a–9e) were also detected, however,
resorcinol (1,3-dihydroxybenzene) (10) or phlorogluci-
pH 6.5 (Schuz et al., 1983), but not at pH 8.0, the opti-
¨
mum pH of the enzyme for chalcone formation from the
physiological starter p-coumaroyl-CoA (Kreuzaler and
Hahlbrock, 1975a,b). Presumably, the pH change signif-
icantly affected charge distribution in the enzyme–sub-
strate and enzyme–intermediate complex, including the
most important charged interactions of the catalytic
triad of Cys164, His303, and Asn336, thus greatly
Fig. 1. TLC based analysis of radiolabeled products of CoA-esters of long-chain fatty acid (7a–7g) by: (a) S. baicalensis CHS and (b) A. hypogaea
STS. Lane a, hexanoyl (C6)-CoA; lane b, octanoyl (C8)-CoA; lane c, decanoyl (C10)-CoA; lane d, dodecanoyl (C12)-CoA; lane e, tetradecanoyl (C14)-
CoA; lane f, hexadecanoyl (C16)-CoA; lane g, octadecanoyl (C18)-CoA; lane h, p-Coumaroyl-CoA.

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I. Abe et al. / Phytochemistry 65 (2004) 2447–2453
influenced the chain elongation reactions of CHS-super-
family enzymes. In the case of the reactions with the
long-chain acyl-CoAs, the triketide pyrone forming
activities of CHS were maximum at pH 8.0 in Tris–
HCl buffer, while those of STS exhibited fairly broad
pH optimum within a range of pH 7.0–9.0 in either
Tris–HCl or potassium phosphate buffer (Fig. 2). On
the other hand, the tetraketide pyrone forming enzyme
activities were slightly higher under acidic pH, which
parallels with the previous observation that most of S.
baicalensis CHS mutants efficiently catalyzed increased
number of condensation reactions under acidic condi-
tion (Abe et al., 2003b).
Interestingly, steady-state kinetic analysis revealed
that the C6, C8, and C10 ester were apparently even bet-
ter starter substrates for both CHS and STS than the
physiological starter p-coumaroyl-CoA (Table 1). In
particular for S. baicalensis CHS, the kcat values of the
C6, C8, and C10 esters (7a–7c) for the ‘‘triketide’’ pyrone
formation were 2.7- to 8.8-fold higher than that of p-
coumaroyl-CoA while the kcat/KM values were 1.2- to
1.9-fold higher than that of the normal substrate. The
enzyme reaction rates were thus significantly increased
when compared with that for the formation of the aro-
matic tetraketides, chalcone or stilbene, from p-couma-
royl-CoA. In contrast, it has been reported that
medium chain aliphatic CoAs (C6–C8) were poor sub-
strates for recombinant M. sativa CHS; the kcat/KM val-
ues were 0.7- to 0.9-fold decreased compared with that
of p-coumaroyl-CoA (Jez et al., 2002). This may suggest
species difference between S. baicalensis (Labiatae) and
M. sativa (Leguminosae) in the active site structure of
the CHS enzyme.
Scheme 3. Enzymatic conversion of long-chain acyl-CoAs (7a–7g) by
recombinant S. baicalensis CHS and A. hypogaea STS. Resorcinol
derivatives (10) and (11) with a newly formed aromatic ring system
were not detected in the reaction mixtures.
It was demonstrated for the first time that the plant
CHS-superfamily enzymes accepted the CoA esters of
Fig. 2. The pH dependence of enzyme activities of: (A) S. baicalensis CHS and (b) A. hypogaea STS when decanoyl (C10)-CoA (7c) was used as a
starter substrate.

I. Abe et al. / Phytochemistry 65 (2004) 2447–2453
2451
Table 1
Steady-state kinetic parameters for enzyme reactionsa
thase (KAS III) that uses long aliphatic chains as sub-
strates (Saxena et al., 2003). Both enzymes share the
Cys-His-Asn catalytic triad as well as the thiolase ababa
overall fold architecture that forms one side of the active
site cavity (Austin and Noel, 2003). Manipulation of the
CHS-superfamily of the type III PKS enzyme reactions
would thus lead to further production of chemically and
structurally diverse unnatural polyketides which is now
in progress in our laboratories.
Starter substrate
Kcat
(minꢀ1
KM
(lM)
kcat/KM
(sꢀ1 Mꢀ1
)
)
S. baicalensis CHS
p-Coumaroyl-CoA
Hexanoyl (C6)-CoA
Octanoyl (C8)-CoA
Decanoyl (C10)-CoA
1.00 0.07
8.82 1.08
3.97 0.24
2.65 0.44
5.17 2.62
24.50 9.87
12.19 1.98
11.04 3.48
3224
6000
5428
4001
A. hypogaea STS
p-Coumaroyl-CoA
Hexanoyl (C6)-CoA
Octanoyl (C8)-CoA
Decanoyl (C10)-CoA
Dodecanoyl (C12)-CoA
a
0.65 0.09
1.01 0.11
2.08 0.11
1.43 0.10
0.50 0.12
5.11 2.14
7.78 2.71
11.70 1.31
4.17 1.05
5.74 0.26
2120
2164
2963
5715
1451
3. Experimental
3.1. Chemicals
Steady-state kinetic parameters were calculated for formation of
major product of the enzyme reaction, i.e. triketide a-pyrones, except
for those of p-coumaroyl-CoA which was for formation of naringenin
chalcone. Lineweaver–Burk plots of data were employed to derive the
apparent KM and kcat values (average of triplicates standard devia-
tion) using EnzFitter software (BIOSOFT).
p-Coumaroyl-CoA was chemically synthesized
according to the published method (Sto¨ckigt and Zenk,
1975). Malonyl-CoA, butyryl-CoA, hexanoyl-CoA,
octanoyl-CoA, decanoyl-CoA, dodecanoyl-CoA, tetra-
decanoyl-CoA, hexadecanoyl-CoA, and octadecanoyl-
CoA were purchased from Sigma. [2-14C]Malonyl-CoA
(48 mCi/mmol) was purchased from Moravek Biochem-
icals (California). 4-Hydroxy-6-pentyl-2-pyrone (8a)
and 4-hydroxy-6-(2-oxoheptyl)-2-pyrone (9a) were ob-
tained in our previous works (Morita et al., 2000, 2001).
long-chain fatty acid (up to C14) as a starter substrate
and carried out sequential decarboxylative condensa-
tions with malonyl-CoA, leading to formation of trike-
tide and tetraketide a-pyrones. The unusually broad
substrate specificities and the catalytic diversities of the
enzymes provided further mechanistic and stereochemi-
cal insights into the polyketide formation reactions of
the type III PKS enzymes. Notably, the results suggested
possible involvement of the CHS-superfamily enzymes
also in the biosynthesis of long-chain alkyl polyphenols
including the palmitoleoyl (C16)-CoA derived urushiol
and ginkgolic acid (anacardic acid), the allergic sub-
stances of lacquer tree (R. verniciflua, Anacardiaceae)
and ginkgo tree (G. biloba, Ginkgoaceae), respectively
(Scheme 2) (Dewick, 2002) although the formation of
these metabolites still needs aromatization of the en-
zyme-bound tetraketide intermediate. None of the
responsible enzymes involved in the biogenesis of the
long-chain alkyl polyphenols have been identified yet.
On the other hand, a novel plant-specific type III PKS
catalyzing formation of olivetolic acid (10a) from a
medium-chain hexanoyl (C6)-CoA as a starter substrate
(Scheme 3) has been recently cloned from Cannabis sati-
va (Cannabinaceae), and regarded as a key enzyme in
the biosynthesis of the cannabinoids (Tanaka et al.,
2001).
3.2. Enzymes
Recombinant S. baicalensis CHS and A. hypogaea
STS with an additional hexahistidine tag at the C-termi-
nal were expressed in Escherichia coli, and purified by
Ni-chelate affinity chromatography as described before
(Abe et al., 2000, 2001, 2002, 2003a,b; Morita et al.,
2000, 2001). Protein concentration was determined by
the Bradford method (Protein Assay, Bio-Rad) with bo-
vine serum albumin as standard.
3.3. Enzyme reaction
The standard reaction mixture contained 27 nmol of
CoA ester of long-chain fatty acid (C4–C18), 54 nmol of
malonyl-CoA, and 5 lg of the purified enzyme in a final
volume of 500 lL of 100 mM potassium phosphate buf-
fer, pH 8.0, containing 1 mM EDTA. Incubations were
carried out at 30 ꢁC for 20 min to overnight, and
stopped by adding 50 lL of 20% HCl. The products
were then extracted with 1 mL of ethyl acetate (·2),
and concentrated by N2 flow. The residue was dissolved
in aliquot of MeOH containing 0.1% TFA, and sepa-
rated by HPLC as described below.
Finally, it should be noted that the active site cavity
of either CHS or STS is not apparently large enough
to accommodate the a-pyrones with the long aliphatic
side chain, suggesting the possibility of an altered bind-
ing pocket as in the case of the recently reported myco-
bacterial type III PKS from M. tuberculosis (Saxena
et al., 2003). Interestingly, Gokhale and co-workers pro-
posed a presence of an additional hydrophobic acyl-
binding pocket based on the structural similarity
between the M. tuberculosis PKS and b-ketoacyl syn-
3.4. Enzyme kinetics
Steady-state kinetic parameters were determined
by using [2-14C]malonyl-CoA (1.8 mCi/mmol) as a
substrate. The experiments were carried out in triplicate

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I. Abe et al. / Phytochemistry 65 (2004) 2447–2453
using five concentrations of long-chain fatty acid (C6,
C8, C10, and C12) in the assay mixture, containing 27
lM of malonyl-CoA, 5 lg of purified enzyme, 1 mM
EDTA, in a final volume of 500 lL of 100 mM Tris–
HCl buffer, pH 8.0. Incubations were carried out at 30
ꢁC for 20 min. The reaction products were extracted
and separated by TLC (Merck Art. 1.11798 Silica gel
60 F254; ethyl acetate/hexane/AcOH = 63:27:5, v/v/v).
Radioactivities were quantified by autoradiography
using a bioimaging analyzer BAS-2000II (FUJIFILM).
Lineweaver–Burk plots of data were employed to derive
the apparent KM and kcat values (average of tripli-
cates standard deviation) using EnzFitter software
(BIOSOFT).
conditions as described above. The ESI capillary tem-
perature and capillary voltage were 225 ꢁC and 3.0 V,
respectively. The tube lens offset was set at 20.0 V. All
spectra were obtained in the negative and positive mode;
over a mass range of m/z 100–500, at a range of one scan
every 2 s. The collision gas was helium, and the relative
collision energy scale was set at 30.0% (1.5 eV).
3.6. Spectroscopic data for the enzyme reaction products
Products from hexanoyl-CoA (7a). Compound 8a:
HPLC: Rt = 31.4 min. LC-ESIMS: MS, m/z 183
[M + H]+, 181 [M ꢀ H]ꢀ, MS/MS (precursor ion at m/
z 181), m/z 137 [M ꢀ H–CO2]ꢀ. UV: kmax 285 nm. Com-
pound 9a: HPLC: Rt = 26.3 min. LC-ESIMS: MS, m/z
225 [M + H]+, 223 [M ꢀ H]ꢀ, MS/MS (precursor ion
at m/z 223), m/z 179 [M ꢀ H–CO2]ꢀ, m/z 125
[C6H5O3]ꢀ. UV: kmax 291 nm. Products from octanoyl-
CoA (7b). Compound 8b: HPLC: Rt = 21.6 min. LC-
ESIMS: MS, m/z 211 [M + H]+, 209 [M ꢀ H]ꢀ, MS/
MS (precursor ion at m/z 209), m/z 165 [M ꢀ H–
CO2]ꢀ. UV: kmax 285 nm. Compound 9b: HPLC:
Rt = 16.1 min. LC-ESIMS: MS, m/z 253 [M + H]+, 251
[M ꢀ H]ꢀ, MS/MS (precursor ion at m/z 251), m/z 207
[M ꢀ H–CO2]ꢀ, m/z 125 [C6H5O3]ꢀ. UV: kmax 291 nm.
Products from decanoyl-CoA (7c). Compound 8c:
HPLC: Rt = 21.7 min. LC-ESIMS: MS, m/z 239
[M + H]+, 237 [M ꢀ H]ꢀ, MS/MS (precursor ion at m/
z 237), m/z 193 [M ꢀ H–CO2]ꢀ. UV: kmax 285 nm. Prod-
ucts from dodecanoyl-CoA (7d). Compound 8d: HPLC:
Rt = 17.7 min. LC-ESIMS: MS, m/z 267 [M + H]+, 265
[M ꢀ H]ꢀ, MS/MS (precursor ion at m/z 265), m/z 221
[M ꢀ H–CO2]ꢀ. UV: kmax 285 nm. Products from tetra-
decanoyl-CoA (7e). Compound 8e: HPLC: Rt = 75.6
min. LC-ESIMS: MS, m/z 295 [M + H]+, 293 [M ꢀ H]ꢀ
MS/MS (precursor ion at m/z 293), m/z 249
[M ꢀ HCO2]ꢀ. UV: kmax 285 nm.
3.5. HPLC and HPLC-ESIMS
The enzyme reaction products were separated by re-
verse phase HPLC (JASCO 880, JASCO) on TSK-gel
ODS-80Ts column (4.6 x 150 mm, TOSOH) with a flow
rate of 0.8 mL/min. Elutions were monitored by a multi-
channel UV detector (MULTI 340, JASCO) at 290, 330
and 360 nm; UV spectra (198–400 nm) were recorded
every 0.4 s. Gradient elution was performed with H2O
(A) and MeOH (B) both containing 0.1% TFA. The gra-
dient profile 1 for the reaction products of hexanoyl-
CoA (7a): 0–5 min, A:B (7:3); 5–17 min, A:B
(7:3 ! 4:6); 17–25 min, A:B (4:6); 25–27 min, A:B
(4:6 ! 3:7). Profile 2 for the reaction products of octa-
noyl-CoA (7b): 0–5 min, A:B (4:6); 5–7 min, A:B
(4:6 ! 3:7); 7–15 min, A:B (3:7); 15–17 min, A:B
(3:7 ! 2:8); 17–25 min, A:B (2:8); 25–27 min, A:B
(2:8 ! 1:9); 27–35 min, A:B (1:9); 35–37 min, A:B
(1:9 ! 0:100); 37–45 min, B(100%). Profile 3 for the
reaction products of decanoyl-CoA (7c): 0–5 min, A:B
(3:7); 5–7 min, A:B (3:7–2:8); 7–15 min, A:B (2:8); 15–
17 min, A:B (2:8 ! 1:9); 17–25 min, A:B (1:9); 25–27
min, A:B (1:9 ! 0:100); 27–45 min, 100% B. Profile 4
for the reaction products of dodecanoyl-CoA (7d): 0–5
min, A:B (2:8); 5–7 min, A:B (2:8 ! 1:9); 7–15 min,
A:B (1:9); 15–17 min, A:B (1:9 ! 0:100); 17–45 min,
100% B. Profile 5 for the reaction products of myris-
toyl-CoA (7e), palmitoyl-CoA (7f), and stearoyl-CoA
(7g): 0–5 min, A:B (7:3); 5–17 min, A:B (7:3 ! 4:6);
17–25 min, A:B (4:6); 25–27 min, A:B (4:6 ! 3:7); 27–
35 min, A:B (3:7); 35–37 min, A:B (3:7 ! 1:3); 37–45
min, A:B (1:3); 45–47 min, A:B (1:3 ! 1:4); 47–55
min, A:B (1:4); 55–57 min, A:B (1:4 ! 1:9); 57–65
min, A:B (1:9); 65–67 min, A:B (1:9 ! 5:95); 67–75
min, A:B (5:95); 75–77 min, A:B (5:95 ! 0:100); 77–85
min, 100% B.
Note added in proof
Recently solved crystal structure of a bacterial type
III PKS revealed a novel cavity extending into the floor
of the active site of the enzyme, providing explanation
for its extra polyketide extension activities when primed
with CoA esters of long-chain fatty acid. (Austin, M. B.,
Izumikawa, M., Bowman, M. E., Udwary, D. W., Fer-
rer, J.-L., Moore, B., Noel, J.P., 2004. Crystal structure
of a bacterial type III polyketide synthase and enzymatic
control of reactive polyketide intermediate. J. Biol.
Chem., published on WEB on August 19.)
On-line HPLC-ESIMS spectra were measured with a
Hewlett–Packard HPLC 1100 series (Wilmington, DE)
coupled to a Finnigan MAT LCQ ion trap mass spec-
trometer (San Jose, CA) fitted with an ESI source.
HPLC separations were carried out under the same
Acknowledgements
The authors are indebted to Professor Joachim
Schroder (Universitat Freiburg) for the A. hypogaea
¨
¨

I. Abe et al. / Phytochemistry 65 (2004) 2447–2453
2453
Jez, J.M., Bowman, M.E., Noel, J.P., 2001. Structure-guided pro-
gramming of polyketide chainlength determination in chalcone
synthase. Biochemistry 40, 14829–14838.
STS clone. This work was in part supported by the 21st
Century COE Program, and Grant-in-Aid for Scientific
Research (Nos. 16510164 and 1531053) from the Minis-
try of Education, Culture, Sports, Science and Technol-
ogy, Japan, and by Grant-in-Aid from the Tokyo
Biochemical Research Foundation, and The Sumitomo
Foundation, Japan.
Jez, J.M., Bowman, M.E., Noel, J.P., 2002. Expanding the biosyn-
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flavanone synthase from cell-suspension cultures of Petroselinum
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