Cloning and functional expression of cDNA encoding aphidicolan-16β-ol synthase: A key enzyme responsible for formation of an unusual diterpene skeleton in biosynthesis of aphidicolin

Full text of DOI:10.1021/ja015747j

5154
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.
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(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.
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R. M.; Croteau, R. B. Biochemistry 2000, 39, 15592-15602.
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10.1021/ja015747j CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/05/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5155
Scheme 2.a Synthesis of syn-Copalyl Diphosphate (3)
tion15 is initiated by formation of an allylic cation followed by
si-face attack of the olefin which is accompanied by a hydride
shift from C-9 to C-8 to afford 8â-pimarenyl cation 10. Electro-
philic attack of the vinyl group and subsequent migration of the
alkyl group give aphidicolenyl cation 11. Final quenching of the
carbocationic intermediate 11 proceeds in two ways: (1) depro-
tonation of either 15-H or 17-H and (2) stereoselective capture
of water. Efficient quench of the cation with water is relatively
unusual in terpene synthases.16 Since the active sites of terpene
synthase are usually hydrophobic to avoid improper quenching
of the cationic intermediate with an external nucleophile such as
water, the stereoselective delivery of water is an interesting
character of this enzyme. P. betae produces a number of diterpene
hydrocarbons including stemar-13-ene.17 Investigation on the
enzyme-product relationship is currently under way.
Despite a remarkable sequence similarity between ACS and
FKS,9 pseudo-enantiotopic and diastereotopic cyclizations of
GGDP 2 with these enzymes afford the 6,6-bicyclic products (+)-
syn-CDP 3 (ACS) and (-)-CDP (FKS), respectively. It suggests
that a relatively small change in amino acid sequence of a cyclase
is enough to promote the formation of a different diterpene carbon
skeleton. Although the sesquiterpene synthases from microorgan-
isms do not share high homology with each other,18 our results
reveal the significant homology among the fungal diterpene
synthases, suggesting that a homology-based PCR strategy could
be useful for cDNA cloning of other fungal diterpene synthases
such as plant terpene synthases. Clustering for the biosynthetic
genes of fungal natural products has been recently recognized.19
This indicates that identification of the aphidicolan-16â-ol syn-
thase gene would allow us to find a gene cluster for the aphidicolin
biosynthesis by chromosome walking.
a Conditions: (a) NaBH4/3 M NaOH, EtOH-CHCl3, 4 °C to room
temperature, 30 min, 92%; (b) DIBAH/THF, -78 °C, 30 min; (c)
Ph3PCH3Br, NaH/DMSO, room temperature to 70 °C, 2 h, 82%, 2 steps;
(d) Ac2O, Et3N, DMAP/toluene, 70 °C, 2 d, 91%; (e) 2,4,6-collidine,
reflux, 8 h, 93%; (f) O2, PdCl2, CuCl/DMF-H2O, room temperature, 5
h, endo:exo ) 4.2:1,14 82%; (g) (EtO)2P(O)CH2CO2Me, NaH/THF, 4 °C
to room temperature, overnight, E:Z )6.4:1, 96%; (h) DIBAH/Et2O, 4
°C to room temperature, 1 h, 99%; (i) ref 13.
Scheme 3. Enzymatic Formation of Aphidicolan-16â-ol (4)
With (GST)-ACS in hand, the intermediacy of syn-copalyl
diphosphate (3) was next examined. Synthesis of 3 was started
from racemic lactone 611 reported by Nishizawa et al. (Scheme
2). Sequential reductions with NaBH4 and DIBAH followed by
Wittig olefination afforded alcohol 7. Dehydration via the
corresponding acetate afforded regioselectively a 4.7:1 mixture
of olefins 8a and 8b. The olefins were converted into 9 by
essentially the same protocol12 (Wacker oxidation, Horner-
Emmons olefination, and DIBAH reduction) reported previously.
After separation of the minor regioisomer derived from 8b by
reverse phase HPLC, 9 was further converted to racemic 3.13
Under conditions identical to those used for incubation of 2, 3
was incubated with (GST)-ACS. GC-MS analysis of the hexane
extract of the reaction mixture showed essentially the same pattern
as that of 2. Thus, the result provided the conclusive evidence
that the enzymatic transformation from 2 to 4 proceeds via
intermediate 3. Coates et al. previously reported that cell-free
conversion of 3 to stemar-13-ene,13 which is structurally related
to 5a and proposed to be a precursor of a phytoalexin in rice.
A proposed mechanism for the reaction catalyzed by aphidi-
colan-16â-ol synthase is shown in Scheme 3. The proton-initiated
type B cyclization15 of 2 provides 3 via an unusual chair-boat
transition state. The second diphosphate-induced type A cycliza-
Acknowledgment. We are grateful to Prof. M. Nishizawa, Tokushima
Bunri University, for providing information on preparation of 6 and Mr.
K. Watanabe and Dr. E. Fukushi in our department for measuring MS
spectra. This work was supported by grants (No. 10556024 to H.O. and
No. 11460053 to T.S.) from the Ministry of Education, Science, Sports
and Culture of Japan.
Supporting Information Available: GC-MS data on the authentic
samples 4, 5a, and 5b and the reaction products of 2 and 3 with (GST)-
ACS, and sequence alignments of ACS and FKS (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA015747J
(14) Selective formation of a byproduct from the exo isomer reduced an
isomer ratio.
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