Enzymatic formation of an unnatural methylated triketide by plant type III polyketide synthases

Article abstract of DOI:10.1016/j.tetlet.2006.10.012

Octaketide synthase, a novel plant-specific type III polyketide synthase from Aloe arborescens, efficiently accepted (2RS)-methylmalonyl-CoA as a sole substrate to produce 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone. On the other hand, a tetraketide-producing chalcone synthase from Scutellaria baicalensis and a diketide-producing benzalacetone synthase from Rheum palmatum also yielded the unnatural methylated C₉ triketide pyrone as a single product by sequential decarboxylative condensations of three molecules of (2RS)-methylmalonyl-CoA.

Full text of DOI:10.1016/j.tetlet.2006.10.012

Tetrahedron Letters 47 (2006) 8727–8730
Enzymatic formation of an unnatural methylated triketide by
plant type III polyketide synthases
Tsuyoshi Abe,a Hisashi Noma,a Hiroshi Noguchia and Ikuro Abea,b,
*
aSchool of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
bPRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
Received 25 September 2006; revised 2 October 2006; accepted 3 October 2006
Available online 20 October 2006
Abstract—Octaketide synthase, a novel plant-specific type III polyketide synthase from Aloe arborescens, efficiently accepted (2RS)-
methylmalonyl-CoA as a sole substrate to produce 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone. On the other hand, a tetraketide-pro-
ducing chalcone synthase from Scutellaria baicalensis and a diketide-producing benzalacetone synthase from Rheum palmatum also
yielded the unnatural methylated C9 triketide pyrone as a single product by sequential decarboxylative condensations of three mole-
cules of (2RS)-methylmalonyl-CoA.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The broad substrate tolerance and catalytic potential of
the chalcone synthase (CHS) (EC 2.3.1.74) superfamily
of type III polyketide synthases (PKSs) are remark-
In previous studies, we have demonstrated that a
tetraketide-producing Scutellaria baicalensis CHS4a
and a diketide-producing Rheum palmatum benzalace-
tone synthase (BAS)6 accepted (2RS)-methylmalonyl-
CoA as an extender substrate to produce unnatural
polyketides.4d,e Further, it has been reported that
Pinus strobus CHS2 carried out a one-step condensa-
tion of a diketide N-acetylcysteaminethioester and
(2RS)-methylmalonyl-CoA to produce a methylated
triketide styrylpyrone.7
able.1,2 Octaketide synthase (OKS) is
a recently
reported novel plant-specific type III PKS from Aloe
arborescens, catalyzing sequential condensation of eight
molecules of malonyl-CoA to produce aromatic C16
octaketides SEK4 and SEK4b (Scheme 1), the longest
polyketides known to be synthesized by the structurally
simple homodimeric type III PKS.3 Like other type III
PKSs,4,5 A. arborescens OKS exhibits unusually broad,
promiscuous substrate specificities; the enzyme accepts
a variety of non-physiological substrates, including
aromatic and aliphatic CoA thioesters, to generate an
array of chemically and structurally distinct unnatural
polyketides.3 Here we now report enzyme reactions
using (2RS)-methylmalonyl-CoA, instead of malonyl-
CoA, as a sole substrate. It was interesting to test
whether the octaketides-producing A. arborescens
OKS accepts (2RS)-methylmalonyl-CoA, with an addi-
tional bulky methyl group, as a starter substrate and
extends an intermediate to produce novel octaketides.
Recombinant A. arborescens OKS was expressed in
E. coli, and purified by Ni-chelate affinity chromato-
graphy as described before.3,8 When incubated with
(2RS)-methylmalonyl-CoA as a sole substrate, A. arbo-
rescens OKS efficiently afforded a single product (UV
kmax 292 nm) (Fig. 1A).9 The LC–ESIMS spectrum
gave a parent ion peak [M+H]+ at m/z 169, suggesting
that the reaction had terminated after two condensa-
tions of (2RS)-methylmalonyl-CoA, and in MS/MS
(precursor ion at m/z 169), the fragment at m/z 125
corresponded to [M+HÀCO2]+, suggesting the
1
presence of an a-pyrone ring. Further, the H NMR
spectrum of the product obtained from a large scale
enzyme reaction (80% yield from 4 mg of (2RS)-meth-
ylmalonyl-CoA) revealed signals at d 2.54 (q, 2H,
J = 7.5 Hz), 1.21 (t, 3H, J = 7.5 Hz), 1.98 (s, 3H),
and 1.96 (s, 3H), indicating the presence of one ethyl
Keywords: Type III polyketide synthase; Chalcone synthase; Octa-
ketide synthase; Benzalacetone synthase; Methylated triketide pyrone.
*
Corresponding author. Tel./fax: +81 54 264 5662; e-mail:
abei@ys7.u-shizuoka-ken.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.10.012

8728
T. Abe et al. / Tetrahedron Letters 47 (2006) 8727–8730
Scheme 1. Proposed mechanism for the formation of (A) naringenin chalcone from 4-coumaroyl-CoA and three molecules of malonyl-CoA by CHS,
(B) benzalacetone from 4-coumaroyl-CoA and one molecule of malonyl-CoA by BAS, and (C) SEK4 and SEK4b from eight molecules of malonyl-
CoA by OKS.
group and two methyl groups. The spectroscopic data
single product by sequential decarboxylative condensa-
tions of three molecules of (2RS)-methylmalonyl-CoA
(Fig. 1B and C).8,9 Here, CHS is a pivotal enzyme for
flavonoid biosynthesis, catalyzing the formation of
naringenin chalcone from 4-coumaroyl-CoA and three
molecules of malonyl-CoA (Scheme 1A), whereas
BAS carries out a one-step decarboxylative condensa-
tion of 4-coumaroyl-CoA with malonyl-CoA to produce
a diketide benzalacetone (Scheme 1B). On the other
hand, it has been reported that a bacterial modular
type I PKS (a cell-free system of recombinant 6-deoxy-
erythronolide B synthase; DEBS 1 + TE) produced
6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone from propion-
yl-CoA, as a starter, and (2RS)-methylmalonyl-CoA,
as an extender, in the absence of NADPH.12 In
this case, (2RS)-methylmalonyl-CoA was not accepted
as a starter substrate by the bacterial modular type I
PKS.
1
(LC–ESIMS, UV, MS, and H NMR)10 were identical
with those of 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyr-
one,11 which was chemically synthesized by condensa-
tion of three molecules of propionyl chloride.11a The
structure of the enzyme reaction product was thus con-
firmed to be 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone,
which was consistent with biogenetic reasoning
(Scheme 2). Interestingly, the compound has been
reported from the fungus Emericella heterothallica,11b
however, it has never been isolated from any plant
sources including A. arborescens.
The C16 octaketides-producing A. arborescens OKS,
normally catalyzing condensation of eight molecules of
malonyl-CoA, thus accepted the bulky (2RS)-methyl-
malonyl-CoA, both as a starter and an extender sub-
strate, and carried out sequential decarboxylative
condensation to produce the methylated C9 triketide
pyrone (Scheme 2). This is the first demonstration of
the enzymatic formation of 6-ethyl-4-hydroxy-3,5-di-
methyl-2-pyrone by the structurally simple plant-specific
type III PKS.
It is remarkable that the plant-specific type III PKSs
accept (2RS)-methylmalonyl-CoA, with an additional
bulky methyl group, both as a starter and an extender
substrate, and catalyze the formation of the non-physio-
logical methylated C9 triketide pyrone. This demon-
strated the further tremendous catalytic potential of
the CHS-superfamily type III PKS enzymes. Manipula-
tion of the functionally divergent type III PKSs by uti-
lizing non-physiological substrates as active-site probes
would thus lead to further production of unnatural
In addition to A. arborescens OKS, a C15 tetraketide-
producing S. baicalensis CHS and a C10 diketide-producing
R. palmatum BAS also accepted (2RS)-methylmal-
onyl-CoA as a starter substrate and efficiently yielded
the unnatural methylated C9 triketide pyrone as a

T. Abe et al. / Tetrahedron Letters 47 (2006) 8727–8730
8729
Scheme 2. Formation of 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone by sequential decarboxylative condensations of three molecules of (2RS)-
methylmalonyl-CoA.
Aid for Scientific Research (Nos. 18510190 and
17310130), Cooperation of Innovative Technology and
Advanced Research in Evolutional Area (CITY AREA,
the Central Shizuoka Area), and the COE21 program
from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
References and notes
1. For recent reviews, see: (a) Schro¨der, J. In Comprehensive
Natural Products Chemistry; Elsevier: Oxford, 1999; Vol. 2,
pp 749–771; (b) Austin, M. B.; Noel, J. P. Nat. Prod. Rep.
2003, 20, 79–110.
2. (a) Ferrer, J. L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.;
Noel, J. P. Nat. Struct. Biol. 1999, 6, 775–784; (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) Tropf, S.; Ka¨rcher, B.; Schro¨der, G.; Schro¨der,
J. J. Biol. Chem. 1995, 270, 7922–7928; (f) Suh, D. Y.;
Fukuma, K.; Kagami, J.; Yamazaki, Y.; Shibuya, M.;
Ebizuka, Y.; Sankawa, U. Biochem. J. 2000, 350, 229–235;
(g) Austin, M. B.; Bowman, M. E.; Ferrer, J.-L.; Schro¨der,
J.; Noel, J. P. Chem. Biol. 2004, 11, 1179–1194; (h) Abe, I.;
Watanabe, T.; Morita, H.; Kohno, T.; Noguchi, H. Org.
Lett. 2006, 8, 499–502.
3. Abe, I.; Oguro, S.; Utsumi, Y.; Sano, Y.; Noguchi, H.
J. Am. Chem. Soc. 2005, 127, 12709–12716.
4. (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; (d) Abe, I.; Takahashi, Y.; Noguchi, H.
Org. Lett. 2002, 4, 3623–3626; (e) Abe, I.; Takahashi, Y.;
Lou, W.; Noguchi, H. Org. Lett. 2003, 5, 1277–1280;
(f) Abe, I.; Watanabe, T.; Noguchi, H. Phytochemistry
2004, 65, 2447–2453; (g) Oguro, S.; Akashi, T.; Ayabe, S.;
Noguchi, H.; Abe, I. Biochem. Biophys. Res. Commun.
2004, 325, 561–567; (h) Abe, I.; Utsumi, Y.; Oguro, S.;
Figure 1. HPLC elution profiles of enzyme reaction products of (A)
Noguchi, H. FEBS Lett. 2004, 562, 171–176; (i) Abe, I.;
A. arborescens OKS, (B) S. baicalensis CHS, and (C) R. palmatum
BAS.
Utsumi, Y.; Oguro, S.; Morita, H.; Sano, Y.; Noguchi, H.
J. Am. Chem. Soc. 2005, 127, 1362–1363; (j) Abe, I.;
Watanabe, T.; Noguchi, H. Proc. Jpn. Acad., Ser. B. 2006,
81, 434–440.
novel poyketides, which is now in progress in our
laboratories.
5. (a) Schuz, R.; Heller, W.; Hahlbrock, K. J. Biol. Chem.
¨
1983, 258, 6730–6734; (b) Zuurbier, K. W. M.; Leser, J.;
Berger, T.; Hofte, A. J. P.; Schro¨der, G.; Verpoorte, R.;
Schro¨der, J. Phytochemistry 1998, 49, 1945–1951; (c) Jez,
J. M.; Bowman, M. E.; Noel, J. P. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 5319–5324; (d) Samappito, S.; Page, J.;
Schmidt, J.; De-Eknamkul, W.; Kutchan, T. M. Planta
2002, 216, 64–71; (e) Samappito, S.; Page, J. E.; Schmidt,
Acknowledgements
This work was supported by the PRESTO program
from Japan Science and Technology Agency, Grant-in-

8730
T. Abe et al. / Tetrahedron Letters 47 (2006) 8727–8730
J.; De-Eknamkul, W.; Kutchan, T. M. Phytochemistry
2003, 62, 313–323.
6. (a) Abe, I.; Takahashi, Y.; Morita, H.; Noguchi, H. Eur. J.
Biochem. 2001, 268, 3354–3359; (b) Abe, I.; Sano, Y.;
Takahashi, Y.; Noguchi, H. J. Biol. Chem. 2003, 278,
25218–25226.
7. 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.
HCl (50 lL). The products were then extracted with ethyl
acetate (1 mL), 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). Gradient elution was
performed with H2O and MeOH, both containing 0.1%
TFA: 0–5 min, 30% MeOH; 5–17 min, linear gradient
from 30% to 60% MeOH; 17–25 min, 60% MeOH; 25–
27 min, linear gradient from 60% to 70% MeOH. For
large-scale enzyme reactions, (2RS)-methylmalonyl-CoA
(4 mg) was incubated with purified recombinant enzyme
(10 mg) in 100 mM phosphate buffer (40 mL, pH 8.0),
containing 1 mM EDTA at 30 ꢁC for 18 h.
8. Recombinant A. arborescens OKS,3 S. baicalensis CHS,4a
and R. palmatum BAS6 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; OKS (95.0 lM and 0.094
minÀ1 for malonyl-CoA), CHS (36.1 lM and 1.26 minÀ1
for 4-coumaroyl-CoA), and BAS (10.0 lM and 1.79 minÀ1
for 4-coumaroyl-CoA).
10. LC–ESIMS: m/z 169 [M+H]+. MS/MS (precursor ion at
1
m/z 169): m/z 125 [M+HÀCO2]+. UV: kmax 292 nm. H
NMR (400 MHz, CD3OD): d 2.54 (q, 2H, J = 7.5 Hz),
1.98 (s, 3H), 1.96 (s, 3H), 1.21 (t, 3H, J = 7.5 Hz). MS
(FAB+): m/z 168 [M]+, 140, 125 [M+HÀCO2]+, 113. The
spectroscopic data were identical with those reported in
the literature.11
9. The reaction mixture contained (2RS)-methylmalonyl-
CoA (108 lM) and the purified recombinant enzyme
(20 lg) in 100 mM potassium phosphate buffer (500 lL,
pH 8.0), containing 1 mM EDTA. Incubations were car-
ried out at 30 ꢁC for 12 h, and stopped by addition of 20%
11. (a) Osman, M. A.; Seibl, J.; Oretsch, E. Helv. Chem. Acta
1977, 60, 3007–3011; (b) Kawahara, N.; Nakajima, S.;
Kawai, K. Phytochemistry 1989, 28, 1546–1548.
12. Pieper, R.; Luo, G.; Cane, D. E.; Khosla, C. J. Am. Chem.
Soc. 1995, 117, 11373–11374.