Alkylresorcylic acid synthesis by type III polyketide synthases from rice Oryza sativa

Article abstract of DOI:10.1016/j.phytochem.2010.02.012

Alkylresorcinols, produced by various plants, bacteria, and fungi, are bioactive compounds possessing beneficial activities for human health, such as anti-cancer activity. In rice, they accumulate in seedlings, contributing to protection against fungi. Alkylresorcylic acids, which are carboxylated forms of alkylresorcinols, are unstable compounds and decarboxylate readily to yield alkylresorcinols. Genome mining of the rice Oryza sativa identified two type III polyketide synthases, named ARAS1 (alkylresorcylic acid synthase) and ARAS2, that catalyze the formation of alkylresorcylic acids. Both enzymes condensed fatty acyl-CoAs with three C₂ units from malonyl-CoA and cyclized the resulting tetraketide intermediates via intramolecular C-2 to C-7 aldol condensation. The alkylresorcylic acids thus produced were released from the enzyme and decarboxylated non-enzymatically to yield alkylresorcinols. This is the first report on a plant type III polyketide synthase that produces tetraketide alkylresorcylic acids as major products.

Full text of DOI:10.1016/j.phytochem.2010.02.012

Phytochemistry 71 (2010) 1059–1067
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Alkylresorcylic acid synthesis by type III polyketide synthases from rice Oryza sativa
*
Miku Matsuzawa, Yohei Katsuyama, Nobutaka Funa, Sueharu Horinouchi
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 May 2009
Received in revised form 8 January 2010
Accepted 9 February 2010
Available online 5 May 2010
Alkylresorcinols, produced by various plants, bacteria, and fungi, are bioactive compounds possessing
beneficial activities for human health, such as anti-cancer activity. In rice, they accumulate in seedlings,
contributing to protection against fungi. Alkylresorcylic acids, which are carboxylated forms of
alkylresorcinols, are unstable compounds and decarboxylate readily to yield alkylresorcinols. Genome
mining of the rice Oryza sativa identified two type III polyketide synthases, named ARAS1 (alkylresorcylic
acid synthase) and ARAS2, that catalyze the formation of alkylresorcylic acids. Both enzymes condensed
fatty acyl-CoAs with three C2 units from malonyl-CoA and cyclized the resulting tetraketide intermedi-
ates via intramolecular C-2 to C-7 aldol condensation. The alkylresorcylic acids thus produced were
released from the enzyme and decarboxylated non-enzymatically to yield alkylresorcinols. This is the
first report on a plant type III polyketide synthase that produces tetraketide alkylresorcylic acids as major
products.
Keywords:
Fatty acid
Oryza sativa
Polyketide synthase
Pyrone
Resorcinol
Resorcylic acid
Ó 2010 Published by Elsevier Ltd.
1. Introduction
type cultivars, accumulate alkylresorcinols, but the Japonica-type
cultivars do not (Suzuki et al., 1996b). They assumed that Japon-
Alkylresorcinols are amphiphilic compounds distributed widely
in plants, bacteria, and fungi (Kozubek and Tyman, 1999). Their
major function in plants is thought to be protection against patho-
genic fungi (García et al., 1997; Cojocaru et al., 1986). In addition,
some health benefits of alkylresorcinols including anti-cancer ef-
fects have also been elucidated (Kozubek and Tyman, 1999; Ross
et al., 2004). Alkylresorcylic acids, carboxylated forms of
alkylresorcinols, rarely occur in the free state except, for example,
in Ginkgo biloba (Gellerman et al., 1976) and Ononis speciosa (Bar-
rero et al., 1989). Suzuki et al. (1996a) identified a mixture of
alkylresorcinols in the etiolated seedling of the Indica-type rice
(Oryza sativa) as antifungal agents. The alkylresorcinol concentra-
tions in the etiolated rice seedlings reached a level comparable to
the ED50 value, which suggested that alkylresorcinols confer resis-
tance against fungi on its seedlings (Suzuki et al., 1996b). They also
found that various types of rice, including the Indica- and Africa-
ica-type rice lost the ability for alkylresorcinol accumulation po-
tential during breeding. Although alkylresorcinols were first
identified and are widely distributed in plants (Kozubek and Ty-
man, 1999), alkylresorcinol synthases in plants have not yet been
identified. Dayan et al. (2007) reported that a root hair extract of
Sorghum bicolor exhibited alkylresorcinol synthase activity, but
the enzyme system responsible for the synthesis was not charac-
terized. Recently, several type III PKSs from plants which catalyze
alkylresorcinol formation, olivetol synthase (OLS) from Cannabis
sativa (Taura et al., 2009), type III PKS involved in sorgoleone bio-
synthesis (Cook et al., 2007) and PpCHS1 from Physcomitrella pat-
ens (Jiang et al., 2008), were described.
Type III PKSs are structurally simple biosynthetic enzymes
found in plants, bacteria, and fungi (Austin and Noel, 2003; Funa
et al., 2006; Ferrer et al., 1999). They consist of small, homodi-
meric, ketosynthase that use CoA esters as substrates and synthe-
size polyketides by using a single catalytic center. Chalcone
synthases (CHSs), which is representative of type III PKSs in plants,
catalyze synthesis of naringenin chalcone by condensing one p-
coumaroyl-CoA with three malonyl-CoAs and catalyzing a C-6 to
C-1 Claisen condensation (Ferrer et al., 1999). Naringenin chalcone
is a common precursor of flavonoids produced by plants. However,
there are many type III PKSs whose starter and extender substrate
specificity, the number of condensed extender substrate, and the
mode of the ring closure reaction, are different. Many of the bioac-
tive polyketides in plants such as stilbenoids and flavonoids are
synthesized by type III PKSs (Austin and Noel, 2003).
Abbreviations: APCIMS, atmospheric pressure chemical ionization mass spec-
trometry; ARAS, alkylresorcylic acid synthase; ArsB, alkylresorcinol synthase B from
Azotobacter vinelandii; CHES, N-cyclohexyl-2-aminoethanesulfonic acid; CUS, curc-
uminoid synthase from rice; EST, expressed sequence tag; HPLC, high-performance
liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/
MS, tandem mass spectrometry; OLS, olivetol synthase; ORAS, 20-oxoalkylresorcylic
acid synthase from Neurospora crassa; PKS, polyketide synthase; SDS–PAGE, SDS–
polyacrylamide gel electrophoresis; SOE, splicing by overlap extension; TLC, thin
layer chromatography.
* Corresponding author. Tel.: +81 3 58415123; fax: +81 3 58418021.
E-mail address: asuhori@mail.ecc.u-tokyo.ac.jp (S. Horinouchi).
0031-9422/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.phytochem.2010.02.012

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M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
In bacteria and fungi, some type III PKSs responsible for alkyl-
non-enzymatically to yield 20-oxoalkylresorcinol. It is expected
that previously reported plant type III PKSs which synthesize
alkylresorcinols catalyzes similar reaction to ArsB as they do not
synthesize alkylresorcylic acids.
This report deals with two type III PKSs in the rice O. sativa,
which synthesize alkylresorcylic acids from fatty acyl-CoAs and
malonyl-CoA. A BLAST search of the Japonica-type rice genome
(Goff et al., 2002) using the CHS sequence in Medicago sativa as
the query, predicted 31 type III PKS candidates. We have continued
to demonstrate the catalytic properties of the enzyme candidates
resorcinol synthesis have been reported. We found that alkylresor-
cinol synthase B (ArsB) in the gram-negative bacterium Azotobacter
vinelandii (Funa et al., 2006), which belongs to the type III PKS
superfamily, catalyzes synthesis of alkylresorcinols. In vitro analy-
sis showed that ArsB releases no alkylresorcylic acids, presumable
intermediates to alkylresorcinols. On the other hand, 20-oxoalkyl-
resorcylic acid synthase (ORAS) in the fungus Neurospora crassa
synthesized 20-oxoalkylresorcylic acid as a major product (Funa
et al., 2007). 20-Oxoalkylresorcylic acids are readily decarboxylated
A
1a: R= n-heneicosyl (C22)
1b: R= n-nonadecyl (C20)
1c: R= n-heptadecyl (C18)
1d: R= n-pentadecyl (C16)
1i : R= n-pentyl (C6)
1e: R= n-tridecyl (C14)
1f : R= n-undecyl (C12)
1g: R= n-nonyl (C10)
1h: R= n-heptyl (C8)
O
1j : R= n-propyl (C4)
1k: R=metyl (C2)
R
S
CoA
1l : R=8-heptadecenyl (C18 : 1 )
starter substrates
2× malonyl-CoA
(Enz)
R
O-
O
S
CoA
R
O
O
i: R= n-pentyl (C6)
2
2a: R= n-heneicosyl (C22)
2b: R= n-nonadecyl (C20)
2c: R= n-heptadecyl (C18)
2d: R= n-pentadecyl (C16)
2e: R= n-tridecyl (C14)
2f : R= n-undecyl (C12)
2g: R= n-nonyl (C10)
2h: R= n-heptyl (C8)
O
O
O
2j: R= n-propyl (C4)
2l: R=8-heptadecenyl
(C18 : 1 )
O
R
S
CoA
triketide intermediates
OH
triketide pyrones
malonyl-CoA
(Enz)
O-
S
CoA
R
O
O
R
i: R= n-pentyl (C6)
3
3a: R= n-heneicosyl (C22)
3b: R= n-nonadecyl (C20)
3c: R= n-heptadecyl (C18)
3d: R= n-pentadecyl (C16)
3e: R= n-tridecyl (C14)
3f : R= n-undecyl (C12)
3g: R= n-nonyl (C10)
3h: R= n-heptyl (C8)
O
7
O
5
O
O
1
1
5
3l: R=8-heptadecenyl
O
O
O
(C18 : 1)
R
S CoA
2
tetraketide intermediates
OH
O
tetraketide pyrones
malonyl-CoA
(Enz)
CoA
5a: R= n-heneicosyl (C22)
5b: R= n-nonadecyl (C20)
OH5c: R= n-heptadecyl (C18)
5d: R= n-pentadecyl (C16)
5e: R= n-tridecyl (C14)
O
O
OH
OH
S
4a: R= n-heneicosyl (C22)
4b: R= n-nonadecyl (C20)
4c: R= n-heptadecyl (C18)
4d: R= n-pentadecyl (C16)
4e: R= n-tridecyl (C14)
R
2
R
O
R
Δ
7
O
O
7
O
O
O
O
5f: R= n-undecyl (C12)
H2O
CO2
5g: R= n-nonyl (C10)
4f: R= n-undecyl (C12)
R
1
S CoA
OH
5h: R= n-heptyl (C8)
O
OH
4l: R=8-heptadecenyl (C18 : 1)
pentaketide intermediates
5l: R=8-heptadecenyl (C18 : 1)
alkylresorcinols
alkylresorcylic acids
7a: R= n-heneicosyl (C22)
7b: R= n-nonadecyl (C20)
(Enz)
O
OH
O
S
CoA
6a: R= n-heneicosyl (C22)
6b: R= n-nonadecyl (C20)
6c: R= n-heptadecyl (C18)
6d: R= n-pentadecyl (C16)
6e: R= n-tridecyl (C14)
7c: R= n-heptadecyl (C18)
OH
R
Δ
2
7d: R= n-pentadecyl (C16)
R
OH
R
O
7
7e: R= n-tridecyl (C14)
7f: R= n-undecyl (C12)
7g: R= n-nonyl (C10)
O
O
O
O
CO2
H2O
6f: R= n-undecyl (C12)
OH
7h: R= n-heptyl (C8)
6l: R=8-heptadecenyl (C18 : 1)
OH
O
7l: R=8-heptadecenyl (C18 : 1)
2′-oxoalkylresorcinols
2′-oxoalkylresorcylic acids
HO
HO
HO
B
STS
C2 C7
2
OH
1
S
CoA
7
5
S
CoA
3× malonyl-CoA
O
O
O
O
O
CO2, H2O
OH
tetraketide intermediate
p-coumaroyl-CoA (8)
resveratrol (12)
HO
Δ
O
OH
OH
CO2
stilbenecarboOxHylic acid (13)
Fig. 1. Summary of the reactions catalyzed by ARAS1 and ARAS2 (A) and STS (B). Non-enzymatic decarboxylation of unstable alkylresorcylic acids (4a–4f, 4l), resulting in the
formation of the corresponding alkylresorcinols (5a–5h, 5l) (A). Products synthesized only by ARAS2 are shown in gray. The main pathway is shown by bold arrows. STS
synthesizes stilbene (12) from one p-coumaroyl-CoA (8) and three malonyl-CoAs (B). Stilbenecarboxylic acid (13) has been shown not to be an intermediate in the STS-
catalyzed reaction (Austin et al., 2004a).

M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
1061
one by one and recently characterized one of these enzymes as a
curcuminoid synthase (CUS) able to synthesize the curcuminoid
scaffold by a mechanism uncommon to the type III PKSs (Katsuy-
ama et al., 2007). Further in vitro analysis of the putative type III
PKSs identified two gene products, Os10g07040 (alkylresorcylic
acid synthase, ARAS1) and Os10g08620 (ARAS2), as enzymes that
catalyzed formation of alkylresorcylic acids by condensing three
malonyl-CoAs with fatty acyl-CoAs and catalyzing C-2 to C-7 intra-
molecular aldol condensation. The resultant alkylresorcylic acids
were non-enzymatically decarboxylated to yield alkylresorcinols.
This is the first report on alkylresorcylic acid synthases from
plants. These results suggest that alkylresorcinols found in a wide
variety of plants might be synthesized from two pathways;
alkylresorcinols might be synthesized by type III PKSs which syn-
thesizes alkylresorcinols, such as olivetol synthase, or via non-
enzymatic decarboxylation of the corresponding alkylresorcylic
acids synthesized by type III PKSs.
ORAS. A homologue of ARAS1 was found in the Indica-type rice
genome whose amino acid identity was 97% (Yu et al., 2002). In
addition, both ARAS1 and ARAS2 have a conserved catalytic triad
composed of Cys-His-Asn, which is crucial for the decarboxylative
condensation activity of all type III PKSs (Austin and Noel, 2003)
(Supplementary Fig. 3).
2.2. In vitro analysis of ARAS1 and ARAS2 reactions
We tested whether ARAS1 and ARAS2 could accept C2 to C22
straight-chain fatty acyl-CoAs (1a–1k) as starter substrates, be-
cause already-known alkylresorcinol synthases like ArsB use vari-
ous long-chain fatty acyl-CoAs as starter substrates (Funa et al.,
2006). Products were analyzed by radio-TLC (Fig. 2) and liquid
chromatography-atmospheric pressure chemical ionization mass
spectrometry (LC–APCIMS) analysis (Supplementary Table 1).
[2-14C]Malonyl-CoA was used for TLC analysis, instead of nonla-
beled malonyl-CoA, as an extender substrate to visualize the reac-
tion products. As
a result, both ARAS1 and ARAS2 yielded
2. Results and discussions
alkylresorcinols (5a–5h), alkylresorcylic acids (4a–4f), triketide
pyrones (2a–2j), and trace amounts of 20-oxoalkylresorcylic acids
(6a–6f), 20-oxoalkylresorcinols (7a–7h), and tetraketide pyrones
2.1. Characterization of ARAS1 and ARAS2
Our BLAST search against the Japonica-type rice genome dat-
abases using the CHS amino acid sequence in M. sativa as the query
(Ferrer et al., 1999) predicted 31 candidate genes that encode a
type III PKS homologue. To characterize the function of these pro-
teins, we carried out in vitro experiments by using recombinant
proteins produced by Escherichia coli and probable substrates, mal-
onyl-CoA as an extender and various CoA esters as a starter. cDNAs
of these type III PKS homologues were cloned by using the method
of splicing by overlap extension (SOE) PCR (Horton et al., 1989) and
overexpressed in E. coli BL21 (DE3) to produce recombinant pro-
teins fused with a His-tag at their N-termini by using the pET sys-
tem. The proteins produced in this way were purified by Ni-NTA
affinity column chromatography to give a major protein band on
SDS–polyacrylamide gel electrophoresis (SDS–PAGE) (Supplemen-
tary Fig. 1). The purified enzymes were then incubated with malo-
nyl-CoA and various starter CoA esters that are known to be used
by type III PKSs. The starter substrates included p-coumaroyl-CoA
(8), cinnamoyl-CoA (9), dihydrocinnamoyl-CoA (10), benzoyl-CoA
(11), and fatty acyl-CoAs (1a–1l). Products were determined by
spectrometric analysis and radio-thin layer chromatography
(TLC) analysis. As a result of this screening assay, two type III
PKS candidates encoded by os10g07040 and os10g08620 were
found to catalyze the formation of alkylresorcylic acids from fatty
acyl-CoAs and malonyl-CoA (Fig. 1A). Therefore, these type III PKSs
were named ARAS1 and ARAS2 (alkylresorcylic acid synthase) from
their functions.
Phylogenetic tree analysis of the 31 type III PKS homologues in
the rice showed that ARAS1 and ARAS2 were in the same clade,
along with os10g08670 and os10g08680 (Supplementary Fig. 2).
Our repeated attempts to obtain the Os10g08670 in the soluble
fraction of E. coli failed, and we were unable to analyze this protein
further. The Os10g08680 protein was predicted to be about 15 kDa
in size from its nucleotide sequence and was smaller than the half
size of other known type III PKSs. Therefore, Os10g08680 would be
a pseudogene.
ARAS1 and ARAS2 shared 84% amino acid identity with each
other. ARAS1 has 49% amino acid identity with the CHS in M. sativa
(Ferrer et al., 1999), 46% with the stilbene synthase (STS) in Pinus
sylvestris (Austin et al., 2004a), 48% with OLS in C. sativa, 14% with
ArsB in A. vinelandii (Funa et al., 2006), and 23% with ORAS in N.
crassa (Funa et al., 2007). ARAS2 shared 49% amino acid identity
with the M. sativa CHS, 47% with the P. sylvestris STS, 48% with
the C. sativa OLS, 14% with A. vinelandii ArsB, and 24% with N. crassa
front
(A) ARAS1
5a
5b
5c
5d
5e
5f
2f
5g
2g
2a
4a
2b
4b
2c
4c
2d
4d
2e
3e
2h
3f
3g
origin
front
m
2
4
6
8
10 12 14 16 18 20 22
(B) ARAS2
5a
5b
5c
5d
5e
5f
2f
5g
2g
5h
2h
2a
4a
2b
4b
2c
4c
2d
4d
2e
3e
2i
3i
2j
3f
3g
3h
origin
m
2
4
6
8
10 12 14 16 18 20 22
Fig. 2. Radio-TLC analysis of the products synthesized by ARAS1 (A) and ARAS2 (B)
from various acyl-CoA starter substrates (1a–1k) and [2-14C]malonyl-CoA. Starter
substrates used were acetyl-CoA (1k) (lane 2), butyryl-CoA (1j) (lane 4), hexanoyl-
CoA (1i) (lane 6), octanoyl-CoA (1h) (lane 8), decanoyl-CoA (1g) (lane 10), lauroyl-
CoA (1f) (lane 12), myristoyl-CoA (1e) (lane 14), palmitoyl-CoA (1d) (lane 16),
stearoyl-CoA (1c) (lane 18), arachidoyl-CoA (1b) (lane 20), and behenoyl-CoA (1a)
(lane 22). Lane m is a control incubation without a starter substrate.

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M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
(3a–3i) from a broad range of CoA esters (1a–1j) (Fig. 2). The differ-
ence between these two enzymes was their preference to the chain
length of the acyl-CoAs. ARAS1 preferred relatively longer chain
fatty acyl-CoAs (1c–1f), where ARAS1 displayed a preference to
CoAs of C12 to C18 (1c–1f), and ARAS2 preferred CoAs of C10 to
C14 (1g–1e).
In addition, we checked whether ARAS1 and ARAS2 could pro-
duce resveratrol (12), which is synthesized from p-coumaroyl-
CoA (8) and malonyl-CoA through the same reaction as in alkylres-
orcinol synthesis; resveratrol (12) is synthesized via tetraketide
formation and its C-2 to C-7 aldol condensation (Fig. 1B). Incuba-
tion of ARAS1 or ARAS2 in the presence of p-coumaroyl-CoA (8)
and malonyl-CoA yielded no products (data not shown). These re-
sults clearly showed that these enzymes are distinct from STS.
There are two types of type III PKSs, represented by ArsB and
ORAS, that are responsible for alkylresorcinol synthesis. Fig. 5
shows presumed synthetic pathways of the resorcinol ring by ArsB
and ORAS (Funa et al., 2007). After condensation of a fatty acyl-CoA
with three malonyl-CoAs, ArsB hydrolyzes the thioester of the tet-
raketide intermediate before aldol condensation, and the decar-
boxylation of C-1 carboxyl group occurs along with aldol
(A) ARAS1
1
10 min
40 min
60 min
4c
3c
[M−H]−=391
5c
15
[M−H] −=347
20
0
5
10
10
10
16
12
8
alkylresorcinol (5c)
triketide pyrone (2c)
A
ARAS1
3c
3c
4c
5c
0
5
15
5c
20
4
4c
0
4
5
6
7
8
9
10
ARAS2
20
15
10
5
alkylresorcinol (5e)
triketide pyrone (2e)
0
15
min
20
(B) ARAS2
2
5 min
4e
4e
3e
3e
3e
0
[M−H]−=335
4
5
6
7
8
9
10
pH
5e
15
[M−H]−=291
20
0
4
alkylresorcinol (5c)
triketide pyrone (2c)
B
20
16
12
8
10
ARAS1
10 min
5e
4
0
10
15
20
0
15
25
35
45
55
4
30 min
4e
5e
alkylresorcinol (5e)
triketide pyrone (2e)
25
ARAS2
20
15
10
5
0
10
15
min
20
Fig. 3. Time course of alkylresorcylic acids (4c and 4e) formation by ARAS1 (A) and
ARAS2 (B). The molecular masses of 3c, 3e, 4c, 4e, 5c, and 5e are 392, 336, 392, 336,
348, and 292, respectively. The black line, representing alkylresorcinols (5c and 5e),
and the red line, representing alkylresorcylic acids (4c and 4e) and tetraketide
pyrones (3c and 3e), indicate extracted ion chromatograms of negative LC–APCIMS
at m/z = 391 (3c), 335 (3e), 391 (4c), 335 (4e), 347 (5c), and 291 (5e). Because of the
instability of 4c and 4e, decarboxylation of 4c and 4e to give 5c and 5e occurred at
the ionization step of mass spectrometry after they had been separated by HPLC
and therefore 4c and 4e are observed in both chromatograms, black and red lines.
Thus, the peaks of alkylresorcinols (5c and 5e) were detected with the peaks of
alkylresorcylic acids (4c and 4e).
0
15
25
35
45
55
temperature (°C)
Fig. 4. The pH dependence (A) and temperature dependence (B) of ARAS1 and
ARAS2. ARAS1 was incubated with stearoyl-CoA (1c) and malonyl-CoA. ARAS2 was
incubated with myristoyl-CoA (1e) and malonyl-CoA. The yields of alkylresorcinols
(5c and 5e) and triketide pyrones (3c and 3e) are plotted in closed circles and open
circles, respectively.

M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
1063
condensation and aromatization. As a result, ArsB mainly produces
alkylresorcinols and does not synthesize alkylresorcylic acids. On
the other hand, ORAS catalyzes aldol condensation and aromatiza-
tion before hydrolysis of the thioester, and therefore the C-1 car-
boxyl group remains in the product. Thus, ORAS synthesizes
alkylresorcylic acids as the direct product. Then alkylresorcylic
acids are readily decarboxylated non-enzymatically and converted
to alkylresorcinols. For elucidation of the reaction mechanism of
ARAS1 and ARAS2, we examined the product profile dependence
on reaction time. In addition, as the long-chain alkylresorcylic
acids were readily decarboxylated at room temperature or by heat,
we kept the quenched reaction product on ice to avoid non-enzy-
matic decarboxylation. Stearoyl-CoA (1c) (for ARAS1) and myri-
stoyl-CoA (1e) (for ARAS2) were used as representative starter
CoAs because they seemed to be the most reactive starter sub-
strates as roughly estimated by the radio-TLC analyses (Fig. 2).
LC–APCIMS analysis of the reaction products showed that alkylres-
orcylic acids were the main products of the ARAS1 and ARAS2 reac-
tions during an early period of the reaction (Fig. 3). However,
prolonged reactions result in a decrease of alkylresorcylic acids
(4c and 4e) and in an increase in the amount of the alkylresorcinols
(5c and 5e). Because of the chemical instability of alkylresorcylic
acids (4c and 4e), decarboxylation of 4c and 4e to give alkylresorci-
nols (5c and 5e) occurred at the ionization step of mass spectrom-
etry after they had been separated by HPLC. We therefore
concluded that the direct products of ARAS1 and ARAS2 were prob-
ably alkylresorcylic acids (4c and 4e). Alkylresorcinols (5c and 5e)
produced by the reaction of these enzymes result from non-enzy-
matic decarboxylation of the corresponding alkylresorcylic acids
(4c and 4e). However, alkylresorcylic acids (4h and 4g) were not
detected when octanoyl-CoAs (1h) and decanoyl-CoA (1g) were
used as starter substrates. These results suggested that ARAS1
and ARAS2 produced alkylresorcinols directly from fatty acyl-CoAs
and malonyl-CoA without synthesizing alkylresorcylic acids when
the acyl chain of the starter substrate is shorter than lauroyl-CoA.
In other words, the reaction mechanism of cyclization might
change when a short-chain fatty acyl-CoAs used as a starter
substrate.
2.3. Enzymatic properties of ARAS1 and ARAS2
Since alkylresorcylic acids were decarboxylated readily to yield
alkylresorcinols during the reaction, extraction and evaporation, it
was rather difficult to quantify the alkylresorcylic acids. Therefore,
we intentionally decarboxylated the products by exposing the
samples to a high temperature, approximately 45 °C, for 30 min
and quantified the alkylresorcinols to analyze the enzymatic prop-
erties of ARAS1 and ARAS2. It is known that b-resorcylic acids can
be decarboxylated non-enzymatically by heat (Clark, 1965). How-
ever, alkylresorcylic acids were less stable and decarboxylated at
lower temperatures than b-resorcylic acid.
We determined the temperature and pH dependence of ARAS1
and ARAS2 for alkylresorcinol synthesis using stearoyl-CoA (1c)
and myristoyl-CoA (1e), respectively. ARAS1 showed a tempera-
ture optimum at 35 °C and pH optimum around 7.0, whereas
ARAS2 showed a temperature optimum at 30 °C and pH optimum
around 6.5 (Fig. 4). Furthermore, the ratio of alkylresorcinol/trike-
tide pyrone production by ARAS1 and ARAS2 differed depending on
pH; the rate of triketide pyrone synthesis increased in alkaline pH.
This indicates that the number of condensation reactions increase
under acidic conditions. A similar catalytic property was also ob-
served for the benzalacetone synthase (BAS) from Rheum palmatum
(Abe et al., 2003) and CUS from O. sativa (Katsuyama et al., 2007).
We measured the kinetic parameters of ARAS1 and ARAS2 for
alkylresorcinols synthesis by varying the concentration of starter
CoAs (1c, 1d, 1e and 1l). We predicted the in vivo substrate utilized
by ARASs according to the composition of alkylresorcinols and
fatty acids in the etiolated rice seedlings of the Indica-type rice
(Suzuki et al., 2003). Extracts from the rice mainly contained
alkylresorcinols (5e, 5d, 5d and 5l) synthesized from myristoyl,
palmitoyl, stearoyl, oleoyl-CoAs (1e, 1d, 1c, and 1l, respectively).
Among them, the alkylresorcinols (4l) from oleoyl-CoA (1l) was
O
S-CoA(Enz)
O
O
S-CoA(Enz)
OH
O
OH
OH
A
aldol condensation,
aromatization
R
R
R
hydrolysis
O
H2O
O
OH
OH
alkylresorcylic acid (4c)
H
O
B
O
O
S-CoA(Enz)
O
decarboxylative
aldol condensation,
aromatization
R
R
OH
R
O
hydrolysis
O
O
H2O, CO2
OH
O
O
O
alkylresorcinol (5c)
O
S-CoA(Enz)
aldol condensation,
S-CoA(Enz)
OH
O
OH
OH
C
R
O
R
R
aromatization
hydrolysis
O
O
O
O
H2O
O
OH
OH
2´-oxoalkylresorcylic acid (6c)
Fig. 5. A proposed mechanism of the reaction catalyzed by ARAS1 and ARAS2 (A), ArsB (B), and ORAS (C). Hydrolysis of the thioester takes place after aldol condensation and
aromatization, and therefore the carboxyl group remains on the product (A and C). Hydrolysis of the thioester takes place firstly, and then decarboxylative aldol condensation
and aromatization take place to form alkylresorcinols (B).

1064
M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
Table 1
Steady-state kinetic parameters of the ARAS1 and ARAS2 reactions.
1e: myristoyl-CoA (C14) 1d: palmitoyl-CoA (C16)
1c: stearoyl-CoA (C18)
1l: oleoyl-CoA (C18: 1)
Km
(l
M)
kcat
kcat/Km
Km
(
lM)
kcat
Kcat/Km
Km
(lM)
kcat
Kcat/Km
Km
(lM)
kcat (ꢁ10ꢀ4 kcat/Km
(ꢁ10ꢀ4 sꢀ1
)
(ꢁsꢀ1 Mꢀ1
)
(ꢁ10ꢀ4 sꢀ1
)
(ꢁsꢀ1 Mꢀ1
)
(ꢁ10ꢀ4 sꢀ1
)
(ꢁsꢀ1 Mꢀ1
)
secꢀ1
)
(ꢁsꢀ1 Mꢀ1
)
ARAS1 10.7 2.41 7.18 0.77 67.4 8.64 18.3 4.23 10.3 2.21 56.3 8.70 9.53 4.01 8.37 1.66 87.9 32.3 17.0 2.21 9.07 0.90 53.4 8.48
ARAS2 106 59.0 240 111 226 15.7 n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d. n. d.
Results are mean S.E. (n = 3). n.d., not determined.
the most abundant component, reaching 50% of the total. However,
the most abundant fatty acid in the etiolated rice seedlings was
palmitic acid (0d). Thus the alkylresorcinol synthase in the rice
ation because alkylresorcylic acids and stilbenecarboxylic acid
(13) were not detected in the reaction products (Funa et al.,
2006; Austin et al., 2004a; Cook et al., 2007; Jiang et al., 2008;
Taura et al., 2009).
could prefer oleoyl-CoA (1l). Since ARAS1 preferred lauroyl-(C12
)
to stearoyl-CoAs (C18) and did not accept short-chain fatty acyl-
CoAs, we assumed that the substrate preference of ARAS1 reflected
the alkylresorcinols composition in vivo.
To clarify whether or not the substrate preference of ARAS1 re-
flects the in vivo alkylresorcinol composition, we measured kinetic
parameters of ARAS1 for alkylresorcinols synthesis from myristoyl,
palmitoyl, stearoyl, oleoyl-CoAs (1e, 1d, 1c, 1l). In the case when
palmitoyl-CoA (1d) as a starter substrate, substrate inhibition
was observed and the activity decreased when substrate concen-
ARAS1 and ARAS2 catalyze a similar cyclization including aldol
condensation with ORAS. However, the former two gave alkylres-
orcylic acids (4a–4f and 4l) as major products, being distinct from
the latter that gave 20-oxoalkylresorcylic (6c) acids as major prod-
ucts. In other words, the difference between ARASs and ORAS is the
number of malonyl-CoA condensation that occur. It is known that
the size of active-site cavity of type III PKSs is important for the
control of the number of malonyl-CoA condensation (Morita
et al., 2007). That is because a larger active-site can keep a longer
polyketide intermediate. Therefore, we assume that ARASs have a
smaller active-site than ORAS.
tration was higher than 8 lM (data not shown). Contrary to our
expectations, however, the kcat/Km value for alkylresorcinol synthe-
sis was highest (88 sꢀ1 Mꢀ1) when C18-CoA (1c) was used, and the
kcat/Km values were not significantly different among myristoyl,
palmitoyl, stearoyl, and oleoyl-CoAs (1e, 1d, 1c, and 1l, respec-
tively) (Table. 1). Kinetic parameters of ARAS2 from myristoyl-
CoA (1e) were estimated in the same way. The kcat/Km value of
ARAS2 was 226 sꢀ1 Mꢀ1, about 2.5-fold higher than that of ARAS1.
Determination of kinetic parameters for triketide pyrone syn-
thesis was hampered because the yields of triketide pyrones were
not detectable under these reaction conditions, in which an excess
amount of malonyl-CoA was contained. This could be interpreted
to mean that the elongation reaction would be promoted when
the concentration of the extender substrate (malonyl-CoA) was
higher than that of starter substrates. Therefore, the ratio of trike-
tide pyrones became much lower than that of the alkylresorcinols
(tetraketides). A similar property was also observed for CUS
(Katsuyama et al., 2007). CUS mainly produces a triketide pyrone,
not a curcuminoid, when the concentration of malonyl-CoA is
higher than that of the starter substrate.
A hydrophobic tunnel, which accounts for binding of long-chain
aliphatic substrates, is present in the structure of PKS18 (Sankar-
anarayanan et al., 2004), ORAS (Goyal et al., 2008), and other type
III PKSs (Austin et al., 2004b), all of which accommodate long-chain
fatty acyl-CoAs as starter substrates. A similar tunnel may also ex-
ist in ARAS1 and ARAS2. ARAS1 formed a resorcinol ring only from
decanoyl to behenoyl-CoAs (1a–1g), and ARAS2 formed it from
octanoyl to behenoyl-CoAs (1a–1h). These results imply that
ARASs catalyze condensation of three malonyl-CoAs and C-2 to
C-7 intramolecular aldol condensation only when an appropriate
fatty acyl-moiety of the starter substrate is accommodated in the
hydrophobic tunnel. In addition, alkylresorcylic acids (4a–4f) were
detected only when lauroyl to behenoyl-CoAs (1a–1f) were used as
starter substrates. Similar results were also observed in analysis of
ORAS (Funa et al., 2007); 20-oxoalkylresorcylic acids (6a, 6b, 6c)
were detected only when acyl-CoAs whose alkyl chains are longer
than stearoyl-CoA (1c) were incorporated. This may be due to a rel-
atively weak interaction of a short acyl-CoA with the putative
hydrophobic tunnel. This weak interaction might change the order
of the reaction. The cleavage of the thioester bond might occur
prior to the C-2 to C-7 aldol condensation in this case. As
os10g08670 belongs to the same clade with in ARAS1 and ARAS2,
the protein encoded by this gene or its ancestor might also catalyze
formation of alkylresorcylic acids.
Although ARAS1 and ARAS2 were very similar in the primary
sequence and catalytic function, their substrate specificities were
slightly different; ARAS2 accepted shorter chain fatty acyl-CoAs
and mainly produced triketide pyrones from them. As in the case
of PKS18 and its mutants (Sankaranarayanan et al., 2004), a slight
difference in the size of the hydrophobic tunnel may result in the
difference in the preferred chain length of the starter CoA.
ARAS1 did not particularly prefer oleoyl-CoA (1l), which is
thought to be used most in vivo. However, the substrate specificity
of ARAS1 regarding the chain lengths of starter-CoAs virtually re-
flects the side-chain length of alkylresorcinols abundant in the eti-
olated seedling of the Indica-type rice. The composition of
alkylresorcinols might be regulated by the substrate specificity of
fatty acid CoA ligase, which catalyzes fatty acyl-CoA formation.
The genome sequence of the Indica-type rice genome shows
that the genome of this type of rice encode 23 type III PKS homo-
2.4. Insight into the activity of ARAS1 and ARAS2
Two type III PKSs found on the Japonica-type rice genome se-
quence, ARAS1 and ARAS2, were established to catalyze the syn-
thesis of alkylresorcylic acids (4a–4f) from long-chain fatty acyl-
CoAs (1a–1f) as starter substrates and malonyl-CoA as an extender
substrate. The mechanism of the resorcinol ring formation by
ARAS1 and ARAS2 seems to resemble that catalyzed by ORAS (Funa
et al., 2007). Thus, the reactions catalyzed by ARAS1 and ARAS2 can
be summarized as shown in Fig. 5. After condensation of one fatty
acyl-CoA and three malonyl-CoAs, the C-2 to C-7 aldol condensa-
tion and aromatization occurs before the thioester bond cleavage
between C-1 and the active site cysteine (or CoA), therefore, the
carboxyl group remains on the product (Fig. 5). Then the alkylres-
orcylic acids produced were released from the enzyme and decar-
boxylated non-enzymatically to yield alkylresorcinols. This
synthetic pathway is crucially different from that catalyzed by
ArsB, STS, OLS, type III PKS involved in sorgoleone biosynthesis
and PpCHS1. The C-2 to C-7 aldol condensation catalyzed by these
enzymes precedes thioester bond cleavage, and the aldol conden-
sation and aromatization that occur accompanied by decarboxyl-

M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
1065
logues (Supplementary Fig. 5). ARAS1 belongs to the same clade
with EAY77763, EAY94420 and EAY94419. EAY77763, EAY94420
and EAY94419 shared 97%, 96% and 94% amino acid identities with
ARAS1 and 85%, 84% and 85% amino acid identities with ARAS2,
respectively. ARAS2 belongs to the same clade with EAY77809
and EAY77810. EAY77809 and EAY77810 shared 75% and 84%
identities with ARAS1 and shared 88% and 85% identities with
ARAS2, respectively. It is highly probable that these homologues
are responsible for the biosynthesis of alkylresorcinols (5c, 5d,
5e, 5l) in the Indica-type rice. Because alkylresorcinols (5c, 5d,
5e, 5l) were not produced at any detectable level by the Japon-
ica-type rice, ARAS1 and ARAS2 in this rice have probably become
inactive during breeding. The expression level of ARAS1 and ARAS2
also suggested this hypothesis. The expression level of ARAS1 and
ARAS2 was estimated by using expressed sequence tag (EST) data-
base (http://rice.plantbiology.msu.edu/). The EST sequence derived
from ARAS1 and ARAS2 had not been obtained from any tissue of O.
sativa, including seedling, flower, sheath, root, immature seed,
stem pistil, endosperm, panicle, root tip, shoot, seed, leaf and cal-
lus. This phenomenon suggested that ARAS1 and ARAS2 are not ex-
pressed in the Japonica-type rice.
CCGGAATTCCATATGCCTGGAGCAGCTACCAC-30 (an EcoRI site is
shown in boldface, an NdeI site is underlined, and the start codon
is shown in italics), ARAS2_1R 50-GATTTTTTACATATCCTCTTCATCTT
GTCC-30, ARAS2_2F 50-AAGAGGATATGTAAAAAATCAGGCATAGA
G-30, and ARAS2_2R 50-CGCGGATCCCTAATTTTGCTTAAGACCAC-30
(a BamHI site is shown in boldface and the stop codon is shown
in italics). The amplified fragments were cloned between the EcoRI
and BamHI sites of pUC19, resulting in pUC19-ARAS1 and pUC19-
ARAS2. The NdeI-BamHI fragments containing ARAS1 and ARAS2
were excised from pUC19-ARAS1 and pUC19-ARAS2, respectively,
and cloned between the NdeI and BamHI sites of pET16b, resulting
in pET16b-ARAS1 and pET16b-ARAS2. For production of His-tagged
ARAS1 and ARAS2, E. coli BL21 (DE3) harboring pET16b-ARAS1 or
pET16b-ARAS2 was grown at 26 °C overnight in Luria broth con-
taining 100 lg/ml ampicillin. The cells were then harvested by
centrifugation and resuspended in 100 mM potassium phosphate
buffer (pH 7.5), 10 mM imidazole and 10% glycerol. After sonica-
tion, cellular debris was removed by centrifugation and the cleared
lysate was applied to a column with a His-bind metal chelation re-
sin (Qiagen, Hilden). The purified His-tagged protein was dialyzed
against 10 mM Tris–HCl (pH 8.0) and 10% glycerol.
At any rate, this is the first demonstration of the plant type III
PKSs that can produce alkylresorcylic acids. Alkylresorcinols, syn-
thesized by decarboxylation of alkylresorcylic acids, are in-born
fungicides of the rice, wheat, maize, barley and other plants. There-
fore, analyzing ARAS homologues in other plants will uncover anti-
fungal defense mechanisms of these plants.
4.3. PKS assay
The standard reaction mixture for radio-thin layer chromatog-
raphy (TLC) assay contained [2-14C]malonyl-CoA (5
l
M), starter
M), 100 mM potassium phosphate buffer (pH7),
and enzyme (50 g) in a total volume of 500 l. The standard reac-
substrate (5
l
l
l
tion mixture for liquid chromatography-atmospheric pressure
chemical ionization mass spectrometry (LC–APCIMS) analysis con-
3. Concluding remarks
tained malonyl-CoA (100
potassium phosphate buffer (pH 7), and 50
volume of 500 l. After the reaction mixture had been preincu-
bated at 30 °C for 2 min, the reactions were initiated by adding
the substrates. The reactions were stopped with addition of 6 M
l
M), starter substrate (100
lM), 100 mM
Two type III PKS, ARAS1 and ARAS2 were cloned from O. sativa
and characterized in vitro. The in vitro analysis of ARAS1 and ARAS2
demonstrated that these enzymes synthesize alkylresorcylic acids
(4a–4f, 4l) from acyl-CoAs (1a–1f, 1l) and malonyl-CoA. This is
the first report describes plant type III PKSs which synthesize alkyl-
resorcylic acids as the major products.
l
g of enzyme in a total
l
HCl (100 ll) after incubation for 20 min, and the products in the
mixture were extracted with EtOAc. The organic layer was sampled
for TLC and LC–APCIMS analyses. Silica gel 60 WF254 thin layer
chromatography plates (Merck) were developed in benzene/ace-
tone/AcOH (85:15:1, v/v/v), and the 14C-labeled compounds were
detected using a Fuji BAS-MS imaging plate (Fuji Film, Tokyo).
LC–APCIMS analysis was carried out by using the Esquire High-
Capacity Trap (HCT) plus system (Bruker Daltonics, Bremen) and
Agilent1100 series (Santa Clara, CA) equipped with a Pegasil-B C4
reversed-phase HPLC column (4.6 ꢁ 250 mm; Senshu Scientific,
Tokyo). The compounds were eluted with a linear gradient [70–
90% CH3CN (solvent A), 60–90% CH3CN (solvent B), 50–90% CH3CN
(solvent C) in H2O (each containing 0.1% AcOH)] over 30 min at a
flow rate of 0.8 ml/min. The triketide pyrone (2a) and alkylresor-
cinol (5a) were identified by comparing their LC–MS and MS/MS
spectra with authentic standards prepared by chemical synthesis
and isolation from an A. vinelandii cell culture (Funa et al., 2006).
Triketide pyrones (2c–2j), tetraketide pyrones (3d–3g), alkylresor-
cylic acids (4c), alkylresorcinols (5b–5g), 20-oxoalkylresorcylic
acids (6b–6c), and 20-oxoalkylresorcinols (7b–7c) were identified
by comparing their LC–MS and MS/MS spectra with those from
authentic standards that had been prepared with ORAS from N.
crassa, as previously described (Funa et al., 2007). Other pyrones
and resorcinols were characterized by comparing the LC–MS and
MS/MS spectra with those of authentic standards prepared with
previously reported ArsB and ArsC (Funa et al., 2006).
4. Experimental
4.1. Materials
E. coli strains JM109 and BL21 (DE3), plasmid pUC19, restriction
enzymes, T4 DNA ligase, and PrimeSTAR DNA polymerase were ob-
tained from Takara Biochemicals (Shiga, Japan), whereas pET16b
was purchased from Novagen (Darmstadt, Germany). [2-14C]Mal-
ony-CoA was purchased from American Radiolabeled Chemicals,
Inc. (St. Louis, USA). p-Coumaroyl-CoA, cinnamoyl-CoA and dihy-
drocinnamoyl-CoA were synthesized by the procedure of Blecher
(1981). Un-labeled malony-CoA, benzoyl-CoA and fatty acyl-CoAs
were purchased from Sigma. The genomic DNA of O. sativa (Nip-
ponbare) was a gift from Y. Tozawa (Ehime University).
4.2. Production and purification of ARAS1 and ARAS2
Splicing by overlap extension (SOE)-PCR (Horton et al., 1989)
was used to obtain ARAS1 and ARAS2 cDNAs. Using the rice genome
DNA as a template, the 0.2-kb exon 1 and 1-kb exon 2 DNA frag-
ments containing the ARAS1 or ARAS2-coding regions were ampli-
fied and connected by PCR with the primers ARAS1_1F 50-CCG
GAATTCCATATGCCTGGAGCAACTACCGC-30 (an EcoRI site is shown
in boldface, an NdeI site is underlined, and the start codon is shown
in italics), ARAS1_1R 50-GATTTATGACATATCCTCTTCATCTTTTCC-30,
ARAS1_2F 50-AAGAGGATATGTCATAAATCTGGTATAGAA-30, ARAS1_2R
50-CGCGGATCCTTAATTTTCCTTCAAACCAC-30 (a BamHI site is shown
in boldface and the stop codon is shown in italics), ARAS2_1F 50-
4.4. pH and temperature dependence analysis
The standard reaction mixture for pH dependence analysis con-
tained [2-14C]malonyl-CoA (5
lM), starter substrate [stearoyl-CoA

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M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
Austin, M.B., Noel, J.P., 2003. The chalcone synthase superfamily of type III
polyketide synthases. Nat. Prod. Rep. 20, 79–110.
Austin, M.B., Bowman, M.E., Ferrer, J.-L., Schröder, J., Noel, J.P., 2004a. An aldol
switch discovered in stilbene synthases mediates cyclization specificity of type
III polyketide synthases. Chem. Biol. 11, 1179–1194.
Austin, M.B., Izumikawa, M., Bowman, M.E., Udwary, D.W., Ferrer, J.-L., Moore, B.S.,
Noel, J.P., 2004b. Crystal structure of a bacterial type III polyketide synthase and
enzymatic control of reactive polyketide intermediates. J. Biol. Chem. 279,
45162–45174.
Barrero, A.F., Sanchez, J.F., Barrón, A., Corrales, F., Rodriguez, I., 1989. Resorcinol
derivatives and other components of Ononis speciosa. Phytochemistry 28, 161–
164.
(1c) for ARAS1, myristoyl-CoA (1e) for ARAS2] (5
potassium phosphate buffer (pH 5.0–9.0) or N-cyclohexyl-2-ami-
noethanesulfonic acid (CHES) buffer (pH 10), and enzyme (10 g)
in a total volume of 100 l. The standard reaction mixture for tem-
perature dependence analysis contained [2-14C]malonyl-CoA
(5 M), starter substrate [stearoyl-CoA (1c) for ARAS1, myristoyl-
CoA (1e) for ARAS2] (5 M), 100 mM potassium phosphate buffer
(pH7 forARAS1, pH6.5 forARAS2), andenzyme(10 g)in a totalvol-
ume of 100 l. After the reaction mixture had been preincubated for
lM), 100 mM
l
l
l
l
l
l
2 min at 30 °C (pH dependence analysis) or 25–50 °C (temperature
dependence analysis), the reactions were initiated by adding the
substrates. The reactions were continued for 20 min. The reactions
were stopped with 6 M HCl (20 ll), and the products in the mixture
were extracted with EtOAc. The organic layer was evaporated, and
kept at 45 °C for 30 min to decarboxylate the alkylresorcylic acids
and the residual material was then dissolved in methanol (10 ll)
for TLC analysis. TLC conditions were as described in Section 4.3.
After separation by TLC, the amounts of polyketides were quantified
by means of [2-14C]malonyl-CoA incorporation.
Blecher, M., 1981. Synthesis of long-chain fatty acyl-coA thioesters using N-
hydroxysuccinimide esters. Methods Enzymol. 72, 404–408.
Clark, L.W., 1965. Further studies on the decarboxylation of b-resorcylic acid in
polar solvents. J. Phys. Chem. 69, 3565–3568.
Cojocaru, M., Droby, S., Glotter, E., Goldman, A., Gottlieb, H.E., Jacoby, B.,
Prusky, D., 1986. 5-(12-heptadecenyl)-resorcinol, the major component of
the antifungal activity in the peel of mango fruit. Phytochemistry 25,
1093–1095.
Cook, D., Dayan, F.E., Rimando, A.M., Nanayakkara, N.P.D., Pan, Z., Duke, S.O.,
Baerson, S.R., 2007. Molecular and biochemical characterization of novel
polyketide synthases likely to be involved in the biosynthesis of sorgoleone.
ACS Symposium Series 955, 141–151.
Dayan, F.E., Watson, S.B., Nanayakkara, N.P.D., 2007. Biosynthesis of lipid
resorcinols and benzoquinones in isolated secretory plant root hairs. J. Exp.
Bot. 58, 3263–3272.
Ferrer, J.-L., Jez, J.M., Bowman, M.E., Dixon, R.A., Noel, J.P., 1999. Structure of
chalcone synthase and the molecular basis of plant polyketide biosynthesis.
Nat. Struct. Biol. 6, 775–784.
Funa, N., Ozawa, H., Hirata, A., Horinouchi, S., 2006. Phenolic lipid synthesis by type
III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.
Proc. Natl. Acad. Sci. USA 103, 6356–6361.
Funa, N., Awakawa, T., Horinouchi, S., 2007. Pentaketide resorcylic acid synthesis by
type III polyketide synthase from Neurospora crassa. J. Biol. Chem. 282, 14476–
14481.
García, S., García, C., Heinzen, H., Moyna, P., 1997. Chemical basis of the resistance of
barley seeds to pathogenic fungi. Phytochemistry 44, 415–418.
Gellerman, J.L., Anderson, W.H., Schlenk, H., 1976. 6-(Pentadec-8-enyl)-2, 4-
dihydroxybenzoic acid from seeds of Ginkgo biloba. Phytochemistry 15, 1959–
1961.
Goff, S.A., Ricke, D., Lan, T.H., Presting, G., Wang, R., Dunn, M., Glazebrook, J.,
Sessions, A., Oeller, P., Varma, H., Hadley, D., Hutchison, D., Martin, C., Katagiri,
F., Lange, B.M., Moughamer, T., Xia, Y., Budworth, P., Zhong, J., Miguel, T.,
Paszkowski, U., Zhang, S., Colbert, M., Sun, W.-L., Chen, L., Cooper, B., Park, S.,
Wood, T.C., Mao, L., Quail, P., Wing, R., Dean, R., Yu, Y., Zharkikh, A., Shen, R.,
Sahasrabudhe, S., Thomas, A., Cannings, R., Gutin, A., Pruss, D., Reid, J., Tavtigian,
S., Mitchell, J., Eldredge, G., Scholl, T., Miller, R.M., Bhatnagar, S., Adey, N.,
Rubano, T., Tusneem, N., Robinson, R., Feldhaus, J., Macalma, T., Oliphant, A.,
Briggs, S., 2002. A draft sequence of the rice genome (Oryza sativa L. ssp.
Japonica). Science 296, 92–100.
Goyal, A., Saxena, P., Rahman, A., Singh, P.K., Kasbekar, D.P., Gokhale, R.S.,
Sankaranarayanan, R., 2008. Structural insights into biosynthesis of
resorcinolic lipids by a type III polyketide synthase in Neurospora crassa. J.
Struct. Biol. 162, 411–421.
Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., Pease, L.R., 1989. Engineering hybrid
genes without the use of restriction enzymes: gene splicing by overlap
extension. Gene 77, 61–68.
Jiang, C., Kim, S.Y., Suh, D.-Y., 2008. Divergent evolution of the thiolase superfamily
and chalcone synthase family. Mol. Phylogenet. Evol. 49, 691–701.
Katsuyama, Y., Matsuzawa, M., Funa, N., Horinouchi, S., 2007. In vitro synthesis of
curcuminoids by type III polyketide synthase from Oryza sativa. J. Biol. Chem.
282, 37702–37709.
Kozubek, A., Tyman, J.H.P., 1999. Resorcinolic lipids, the natural non-isoprenoid
phenolic amphiphiles and their biological activity. Chem. Rev. 99, 1–25.
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam,
H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J.,
Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23,
2947–2948.
4.5. Determination of kinetic parameters
The standard reaction mixture contained starter substrate,
[2-14C]malonyl-CoA (61,700 dpm) (100
l
M), 100 mM potassium
phosphate buffer (pH 7 for ARAS1, pH 6.5 for ARAS2), and enzyme
(4 g) in a total volume of 100 l. The concentrations of starter
substrates were varied between 1.0 and 7.0 M for palmitoyl-
CoA (1d), stearoyl-CoA (1c), and oleoyl-CoA (1l), and 1.5–7.0
for myristoyl-CoA (1e) when incubated with ARAS1 and 15–
50 M for myristoyl-CoA (1e) when incubated with ARAS2, respec-
l
l
l
lM
l
tively. The reaction mixtures containing ARAS1 were incubated at
35 °C and containing ARAS2 were incubated at 30 °C. After 2 min
incubation, reaction was initiated by adding substrates, starter
substrates and malonyl-CoA. After 20 min incubation, reaction
was quenched by adding 6 M HCl (20 ll). Reaction products were
extracted by EtOAc and evaporated to dryness. The reaction prod-
ucts were kept at 45 °C for 30 min to enhance decarboxylation of
the alkylresorcylic acids. The reaction and TLC conditions were as
described in Section 4.3. After separation by TLC, the amounts of
polyketides were quantified by means of [2-14C]malonyl-CoA
incorporation. Kinetic parameters were deduced by nonlinear
least-squares fitting to the Michaelis–Menten equation. Linewe-
aver–Burk plot was exceptionally used for determination of kinetic
parameters for palmitoyl-CoA (1d) because fitting to the Michae-
lis–Menten equation was unsuitable due to substrate inhibition
occurring in a linear region.
Acknowledgments
We thank Y. Tozawa (Ehime University) for providing us with the
O. sativa genomic DNA. This work was supported by a research grant
from the New Energy and Industrial Technology Development Orga-
nization of Japan and a Grant-in-Aid for Scientific Research on Prior-
ity Area ‘‘Applied Genomics” from Monkasho. Y. katsuyama was
supported by the Japan Society for Promotion of Science.
Morita, H., Kondo, S., Oguro, S., Noguchi, H., Sugio, S., Abe, I., Kohno, T., 2007.
Structural insight into chain-length control and product specificity of
pentaketide chromone synthase from Aloe arborescens. Chem. Biol. 14, 359–
369.
Ross, A.B., Kamal-Eldin, A., Åman, P., 2004. Dietary alkylresorcinols: absorption,
bioactivities, and possible use as biomarkers of whole-grain wheat- and rye-
rich foods. Nutr. Rev. 62, 81–95.
Appendix A. Supplementary data
Sankaranarayanan, R., Saxena, P., Marathe, U.B., Gokhale, R.S., Shanmugam, V.M.,
Rukmini, R., 2004. A novel tunnel in mycobacterial type III polyketide synthase
reveals the structural basis for generating diverse metabolites. Nat. Struct. Mol.
Biol. 11, 894–900.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2010.02.012.
Suzuki, Y., Esumi, Y., Hyakutake, H., Kono, Y., Sakurai, A., 1996a. Isolation of 5-(80Z-
heptadecenyl)-resorcinol from etiolated rice seedlings as an antifungal agent.
Phytochemistry 41, 1485–1489.
References
Suzuki, Y., Saitoh, C., Hyakutake, H., Kono, Y., Sakurai, A., 1996b. Specific
accumulation of antifungal 5-alk(en)ylresorchinol homologs in etiolated rice
seedlings. Biosci. Biotechnol. Biochem. 60, 1786–1789.
Abe, I., Sano, Y., Takahashi, Y., Noguchi, H., 2003. Site-directed mutagenesis of
benzalacetone synthase. The role of the Phe215 in plant type III polyketide
synthases. J. Biol. Chem. 278, 25218–25226.

M. Matsuzawa et al. / Phytochemistry 71 (2010) 1059–1067
1067
Suzuki, Y., Kurano, M., Esumi, Y., Yamaguchi, I., Doi, Y., 2003. Biosynthesis of 5-
alkylresorcinol in rice: incorporation of a putative fatty acid unit in the 5-
alkylresorcinol carbon chain. Bioorg. Chem. 31, 437–452.
Taura, F., Tanaka, S., Taguchi, C., Fukamizu, T., Tanaka, H., Shoyama, Y., Morimoto, S.,
2009. Characterization of olivetol synthase, a polyketide synthase putatively
involved in cannabinoid biosynthetic pathway. FEBS Lett. 583, 2061–2066.
Yu, J., Hu, S., Wang, J., Wong, G.K., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X.,
Cao, M., Liu, J., Sun, J., Tang, J., Chen, Y., Huang, X., Lin, W., Ye, C., Tong, W., Cong,
L., Geng, J., Han, Y., Li, L., Li, W., Hu, G., Huang, X., Li, W., Li, J., Liu, Z., Li, L., Liu, J.,
Qi, Q., Liu, J., Li, L., Li, T., Wang, X., Lu, H., Wu, T., Zhu, M., Ni, P., Han, H., Dong, W.,
Ren, X., Feng, X., Cui, P., Li, X., Wang, H., Xu, X., Zhai, W., Xu, Z., Zhang, J., He, S.,
Zhang, J., Xu, J., Zhang, K., Zheng, X., Dong, J., Zeng, W., Tao, L., Ye, J., Tan, J., Ren,
X., Chen, X., He, J., Liu, D., Tian, W., Tian, C., Xia, H., Bao, Q., Li, G., Gao, H., Cao, T.,
Wang, J., Zhao, W., Li, P., Chen, W., Wang, X., Zhang, Y., Hu, J., Wang, J., Liu, S.,
Yang, J., Zhang, G., Xiong, Y., Li, Z., Mao, L., Zhou, C., Zhu, Z., Chen, R., Hao, B.,
Zheng, W., Chen, S., Guo, W., Li, G., Liu, S., Tao, M., Wang, J., Zhu, L., Yuan, L.,
Yang, H., 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. indica).
Science 296, 79–92.