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J. Am. Chem. Soc. 2001, 123, 5154-5155
Cloning and Functional Expression of cDNA
Encoding Aphidicolan-16â-ol Synthase: A Key
Enzyme Responsible for Formation of an Unusual
Diterpene Skeleton in Biosynthesis of Aphidicolin
Hideaki Oikawa,*,† Tomonobu Toyomasu,§ Hiroaki Toshima,†
Satoshi Ohashi,† Hiroshi Kawaide,‡ Yuji Kamiya,‡
Minoru Ohtsuka,§ Shoko Shinoda,§ Wataru Mitsuhashi,§ and
Takeshi Sassa*,§
Department of Applied Bioscience
Graduate School of Agriculture
Hokkaido UniVersity, Sapporo 060-8589, Japan
Institute of Physical and Chemical Research (RIKEN)
Wako, Saitama 351-0198, Japan
Figure 1. GC chart of the products of incubation of 2 with (GST)-ACS.
DB-1 capillary column (φ 0.25 mm × 30 m, J&W Scientific); 100-280
°C, 5 °C/min. Numbers on the top of peaks correspond to compound
numbers in the text.
Department of Bioresource Engineering
Faculty of Agriculture, Yamagata UniVersity
Tsuruoka, Yamagata 997-8555, Japan
Scheme 1. The Biosynthetic Pathway of Aphidicolin (1)
ReceiVed March 2, 2001
ReVised Manuscript ReceiVed April 23, 2001
A tetracyclic diterpene, aphidicolin (1), was first isolated as
an antiviral agent against Herpes simplex type 1.1 Later, it was
found that 1 shows a variety of biological activity such as
antitumor2a and phytotoxic2b and specific inhibition of DNA
polymerase R.2c Because of this latter property, 1 is a com-
mercially available agent for studying cell cycles. Recently, it
has been reported that 1 specifically damages a fragile site of the
manmalian genome.2d Besides its remarkable bioactivity, its
unique molecular skeleton has attracted synthetic chemists. This
prompted numerous synthetic studies and, to date, more than 10
groups have achieved the total synthesis of 1.3
Based on an incorporation study with doubly isotope labeled
precursors, Bu’Lock et al. proposed that the molecular skeleton
of 1 is constructed by a stepwise cyclization of geranylgeranyl
diphosphate (GGDP, 2) to aphidicol-16-ene (5a) via an unusual
intermediate syn-copalyl diphosphate (syn-CDP, 3) as outlined
in Scheme 1.4 According to incorporation studies with the
plausible intermediates, Hanson et al. established that post-
cyclization conversion to 1 occurs by two routes: a major route
via aphidicolan-16â-ol (4) and a minor route via 5a.5 Accumula-
tion of less oxidized intermediates in mycelia treated with P-450
inhibitors led us to propose cytochrome P-450 dependent sequen-
tial hydroxylations from 4 to 1.6 These data indicate that 4 is a
major cyclization product of the corresponding diterpene cyclase.
Herein, we report the cDNA cloning and functional expression
of aphidicolan-16â-ol synthase that is a key enzyme in aphidicolin
biosynthesis in the fungus Phoma betae PS-13.2b
Reverse transcription-polymerase chain reaction (RT-PCR) with
mRNA from P. betae and degenerate primers7 based on the
conserved amino acid sequences of plant and fungal diterpene
cyclases allowed us to amplify the 1100-bp band that showed a
significant similarity to fungal ent-kaurene synthases (FKS).7 The
nucleotide sequence of the full-length cDNA was determined by
5′ rapid amplification of the cDNA ends (5′-RACE) and 3′-RACE
by using gene-specific primers. This contained the predicted 2997-
bp open reading frame, encoding a product of 998 amino acids
that was named aphidicolan-16â-ol synthase (ACS).8 Homology
searches indicate that the derived amino acid sequence of ACS
shows good identity (36-37%)9 with FKS and contains aspartate/
glutamate rich motifs (DXDD and DD(E)XXD(E)). A full-length
cDNA was ligated into a pGEX 4T-3 vector for a protein-
expression analysis and the glutathione S-transferase (GST)-ACS
fusion protein was expressed in Escherichia coli JM109. Purifica-
tion of a cell-free extract with affinity chromatography for GST
gave reasonably pure (GST)-ACS. The hexane extracts of the
reaction mixture obtained by incubation of 2 with (GST)-ACS
afforded three products 4, 5a, and 5b (Figure 1). These products
of the (GST)-ACS were identified, by comparison of retention
time and mass spectra with those of synthetic standards,6b as
aphidicolan-16â-ol (4, 87%), aphidicol-16-ene (5a, 5%), and
aphidicol-15-ene (5b, 8%). Since all products are found in the
mycelial extracts of P. betae, it is confirmed that these products
are produced by a single enzyme. Recently, Croteau et al. reported
that abietadiene synthase also produces multiple products.10
* Address correcpondence to these authors. E-mail: hoik@
chem.agr.hokudai.ac.jp and tsassa@tds1.tr. yamagata-u.ac.jp.
† Hokkaido University.
§ Yamagata University.
‡ RIKEN.
(1) Dalziel, W.; Hesp, B.; Stevenson, K. M.; Jarvis, J. A. J. J. Chem. Soc.,
Perkin Trans. 1 1973, 2841-2851.
(2) (a) Douros, J.; Suffness, M. New Anticancer Drugs; Carter, S., Sakurai,
Y., Eds.; Springer-Verlag: Berlin, 1980. (b) Ichihara, A.; Oikawa, H.; Hayashi,
K.; Hashimoto, M.; Sakamura, S.; Sakai, R. Agric. Biol. Chem. 1984, 48,
1687-1689. (c) Ikegami, S.; Taguchi, T.; Ohashi, M.; Oguro, M.; Nagano,
H.; Mano, Y. Nature 1978, 275, 458-460. (d) Palin, A. H.; Critcher, R.;
Fitzgerald, D. J.; Anderson, J. N.; Farr, C. J. J. Cell Sci. 1998, 111, 1623-
1634.
(3) For a review on the synthesis of aphidicolanes, see: Toyota, M.; Ihara,
M. Tetrahedron 1999, 55, 5641-5679.
(4) Adams, M. R.; Bu’Lock, J. D. J. Chem. Soc., Chem. Commun. 1975,
389-391.
(7) (a) Kawaide, H.; Imai, R.; Sassa, T.; Kamiya, Y. J. Biol. Chem. 1997,
272, 21706-21712. (b) Toyomasu, T.; Kawaide, H.; Ishizaki, A.; Shinoda,
S.; Otsuka, M.; Mitsuhashi, W.; Sassa, T. Biosci. Biotechnol. Biochem. 2000,
64, 660-664.
(8) The nucleotide sequence reported in this paper was deposited in the
GenBank/EBI Data Bank with accession number AB049075.
(9) See Supporting Information for sequence alignments for ACS and FKS.
(10) Peters, R. J.; Flory, J. E.; Jetter, R.; Ravn, M. M.; Lee, H.-J.; Coates,
R. M.; Croteau, R. B. Biochemistry 2000, 39, 15592-15602.
(5) (a) Ackland, M. J.; Hanson, J. R.; Yeoh, B. L.; Ratcliffe, A. H. J. Chem.
Soc., Perkin Trans. 1 1985, 2705-2707. (b) Ackland, M. J.; Gordon, J. F.;
Hanson, J. R.; Yeoh, B. L.; Ratcliffe, A. H. J. Chem. Soc., Perkin Trans. 1
1988, 1477-1480.
(6) (a) Oikawa, H.; Ichihara, A.; Sakamura, S. Agric. Biol. Chem. 1989,
53, 299-300. (b) Oikawa, H.; Ohashi, S.; Ichihara, A.; Sakamura, S.
Tetrahedron 1999, 55, 7541-7554.
10.1021/ja015747j CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/05/2001