A short-chain dehydrogenase involved in terpene metabolism from Zingiber zerumbet

Article abstract of DOI:10.1111/j.1742-4658.2011.08211.x

The rhizome oil of Zingiber zerumbet Smith contains an exceptionally high content of sesquiterpenoids with zerumbone, a predominating potential multi-anticancer agent. Biosynthetic pathways of zerumbone have been proposed, and two genes ZSS1 and CYP71BA1 that encode the enzymes catalyzing the first two steps have been cloned. In this paper, we isolated a cDNA clone (ZSD1) that encodes an alcohol dehydrogenase capable of catalyzing the final step of zerumbone biosynthesis. ZSD1 has an open reading frame of 804 bp that encodes a 267-residue enzyme with a calculated molecular mass of 28.7 kDa. After expression in Escherichia coli, the recombinant enzyme was found to catalyze 8-hydroxy-α-humulene into zerumbone. ZSD1 is a member of the short-chain dehydrogenase/reductase superfamily (SDR) and shares high identities with other plant SDRs involved in secondary metabolism, stress responses and phytosteroid biosynthesis. In contrast to the transcripts of ZSS1 and CYP71BA1, which are almost exclusively expressed in rhizomes, ZSD1 transcripts are detected in leaves, stems and rhizomes, suggesting that ZSD1 may also be involved in other biological processes. Consistent with its proposed flexible substrate-binding pocket, ZSD1 also converts borneol to camphor with K_m and k _cat values of 22.8 μm and 4.1 s^-1, displaying its bisubstrate feature.

Full text of DOI:10.1111/j.1742-4658.2011.08211.x

A short-chain dehydrogenase involved in terpene
metabolism from Zingiber zerumbet
1
,
1,
2
3
4
Sho Okamoto *, Fengnian Yu *, Hisashi Harada , Toshihide Okajima , Jun-ichiro Hattan ,
2
,4
1
Norihiko Misawa and Ryutaro Utsumi
1
2
3
4
Department of Bioscience, Graduate School of Agriculture, Kinki University, Nara, Japan
Central Laboratories for Frontier Technology, Kirin Holdings Co. Ltd., Ishikawa, Japan
Institute of Scientific and Industrial Research, Osaka University, Japan
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Japan
Keywords
The rhizome oil of Zingiber zerumbet Smith contains an exceptionally high
content of sesquiterpenoids with zerumbone, a predominating potential
multi-anticancer agent. Biosynthetic pathways of zerumbone have been
proposed, and two genes ZSS1 and CYP71BA1 that encode the enzymes
catalyzing the first two steps have been cloned. In this paper, we isolated a
cDNA clone (ZSD1) that encodes an alcohol dehydrogenase capable of
catalyzing the final step of zerumbone biosynthesis. ZSD1 has an open
reading frame of 804 bp that encodes a 267-residue enzyme with a calcu-
lated molecular mass of 28.7 kDa. After expression in Escherichia coli, the
recombinant enzyme was found to catalyze 8-hydroxy-a-humulene into ze-
rumbone. ZSD1 is a member of the short-chain dehydrogenase ⁄ reductase
superfamily (SDR) and shares high identities with other plant SDRs
involved in secondary metabolism, stress responses and phytosteroid bio-
synthesis. In contrast to the transcripts of ZSS1 and CYP71BA1, which
are almost exclusively expressed in rhizomes, ZSD1 transcripts are detected
in leaves, stems and rhizomes, suggesting that ZSD1 may also be involved
in other biological processes. Consistent with its proposed flexible sub-
bisubstrate; short-chain dehydrogenase-
reductase; zerumbone; Zingiber zerumbet
Smith; ZSD1
Correspondence
R. Utsumi, Department of Bioscience,
Graduate School of Agriculture, Kinki
University, Nakamachi, Nara 631-8505,
Japan
Fax: +81 742 43 8976
Tel: +81 742 43 7306
E-mail: utsumi@nara.kindai.ac.jp
*
These authors contributed equally to this
work
(
Received 27 February 2011, revised 20
May 2011, accepted 7 June 2011)
strate-binding pocket, ZSD1 also converts borneol to camphor with K
1
m
doi:10.1111/j.1742-4658.2011.08211.x
)
and kcat values of 22.8 lM and 4.1 s , displaying its bisubstrate feature.
Introduction
Zingiber zerumbet Smith, a member of the Zingibera-
ceae family, is widely cultivated for its medicinal pro-
perties throughout the tropics and the subtropics,
particularly in Southeast Asia. The rhizome of
Z. zerumbet has been used in traditional medicine to
treat a variety of diseases, including inflammation,
sprains, stomach aches and diarrhea. Its special aro-
matic odor is due to the complex mixture of the monot-
erpenoids and sesquiterpenoids in its rhizome essential
oil. Although the composition of the rhizome oil of
Z. zerumbet shows variations among different ecotypes
[1–5], the most abundant and characteristic component
is zerumbone, a pharmaceutically interesting sesquiterp-
enoid. In recent decades, zerumbone has attracted inten-
sive attention for its anti-inflammatory [6] and multiple
anticancer properties [7–11].
It has been assumed that the biosynthetic pathway
of zerumbone in Z. zerumbet from the common sesqui-
terpene precursor farnesyl diphosphate is analogous to
the biosynthesis of other plant terpene ketones [12,13]
Abbreviations
GatDH, galactitol dehydrogenase; 8-HAH, 8-hydroxy-a-humulene; SDR, short-chain dehydrogenase ⁄ reductase.
2
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S. Okamoto et al.
An SDR involved in terpene metabolism
(Fig. 1). Two genes, ZSS1 and CYP71BA1, encoding
Results and Discussion
a-humulene synthase and a-humulene-8-hydoxylase,
respectively, which are proposed to be responsible for
the two early committed steps of zerumbone biosyn-
thesis, have been cloned and characterized [14,15]. The
final step was assumed to be catalyzed by a short-chain
dehydrogenase.
Homology-based cloning of ZSD1 encoding a
dehydrogenase from rhizomes of Z. zerumbet
Plant SDRs are one of the most important terpene-
modifying enzymes that catalyze oxidation ⁄reduction
reactions [21–24]. Although there has been increasing
evidence that a multifunctional cytochrome P450 can
catalyze the successive conversion of terpene hydrocar-
bons to the corresponding ketones [25,26], our previ-
ous studies showed that CYP71BA1 cannot catalyze
the successive reactions from a-humulene to zerum-
bone [15]. Therefore, we assumed that the final step is
the oxidation of 8-HAH to zerumbone by an alcohol
dehydrogenase.
Short-chain dehydrogenases ⁄ reductases (SDRs) are
highly divergent NAD(P)(H)-dependent enzymes with
typically about 250 amino acid residues [16]. They
cover a wide range of substrate spectra, ranging from
alcohols, steroids, sugars and aromatic compounds to
xenobiotics [17]. In plants, SDRs participate in many
important biochemical and physiological processes,
such as secondary metabolism [18], stress responses
[
19] and phytosteroid biosynthesis [20]. In terpenoid
In an effort to isolate dehydrogenase cDNA from
Z. zerumbet, RT-PCR was conducted using two degen-
erate primers designed on the highly conserved regions
of plant SDRs (Fig. 2), and a predominant fragment
with the predicated length of approximately 350 bp
was generated. After sequencing, this fragment showed
a high similarity to known SDRs. Then, a combination
of 5¢RACE and 3¢RACE was performed using specific
primers to obtain the remaining 5¢ and 3¢ sequences.
Two different types of full-length cDNA clones were
obtained. One of them, named ZSD1, has an ORF of
metabolism, SDRs are one of the most important ter-
pene-modifying enzymes for catalyzing oxida-
tion⁄ reduction reactions, which are crucial for the
synthesis of biologically active terpenoid molecules. In
mint, two similar short-chain dehydrogenases and two
reductases involved in monoterpenoid biosynthesis
have been reported [21,22]. Recently, a multisubstrate
monoterpene alcohol dehydrogenase and a terpenoid
reductase have also been characterized from Artemi-
sia annua [23,24]. To elucidate the final step of the
zerumbone biosynthesis, a homology-based cloning
strategy was employed to clone short-chain alcohol
dehydrogenase genes from Z. zerumbet rhizomes. Here,
we describe the isolation and the functional character-
ization of a cDNA clone encoding an alcohol dehydro-
genase that can convert both 8-hydroxy-a-humulene
8
04 bp that encodes a 267-residue enzyme with a cal-
culated molecular mass of approximately 28.7 kDa
and an isoelectric point of 5.63 calculated from the
deduced amino acid sequence (Fig. S1) (accession no.
+
AB480831). ZSD1 contains a NAD -binding motif
(
(
TGxxxGxG),
a
downstream structural domain
(8-HAH) to zerumbone and monoterpene alcohol bor-
neol to camphor.
(N ⁄C)NAG) of undefined function and an active site
sequence (YxxxK), indicating that it belongs to the
classical’ subfamily of the SDR superfamily [16]
Fig. S1). A blast search of the GenBank database
‘
(
demonstrated that ZSD1 is most closely related to sev-
eral putative alcohol dehydrogenases of other plant
species with the highest identity 65%. Among the
functionally characterized enzymes, ZSD1 is shown to
be highly similar to SDRs involved in abscisic acid
biosynthesis with identities of 61% (Citrus sinensis),
5
9% (Solanum tuberosum) and 56% (Arabidopsis thali-
ana). Interestingly, significant homology is also
observed with Digitalis lanata 3-b-hydroxysteroid
dehydrogenase (48% identity) [20]. However, phyloge-
netic analysis revealed its distant relationship to sev-
eral known terpene-modifying SDRs, such as
Mentha · piperita isopiperitenol dehydrogenase (35%
identity), its menthol dehydrogenase (14% identity)
[27] and Aedes aegypti farnesol dehydrogenase (25%
identity) [28] (Fig. 2).
Fig. 1. Proposed pathway for zerumbone biosynthesis in
Z. zerumbet.
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An SDR involved in terpene metabolism
S. Okamoto et al.
ZSD1 transcript accumulation is detected in
leaves, stems and rhizomes
The total RNA from leaf, stem and rhizome tissues of
Z. zerumbet was extracted, and quantitative real-time
PCR analysis was performed to examine the mRNA
levels of ZSD1 in different tissues. A partial cDNA
sequence of ubiquitin was used as an internal reference.
The results showed that ZSD1 is highly expressed in all
of the tissues examined (Fig. 4A). This finding is sur-
prising in that zerumbone is only present in rhizomes of
Z. zerumbet, and transcripts of ZSS1 and CYP71BA1
encoding the two upstream enzymes of zerumbone
biosynthesis are almost exclusively accumulated in rhi-
zomes (Fig. 4B,C) [15]. Thus, in addition to converting
8-HAH to zerumbone, ZSD1 may also contribute to the
dehydrogenation reactions of other compounds as well.
ZSD1 also converts borneol to camphor in vitro
Fig. 2. Phylogenetic analysis of ZSD1 and related functionally char-
acterized SDRs. The tree was constructed using the neighbor-join-
ing algorithm with CLUSTALW (http://align.genome.jp/) and visualized
with TREEVIEW. Sequences and associated GenBank accession num-
bers are Forsythia · intermedia secoisolariciresinol dehydrogenase
To explore the possibility that ZSD1 also participates
in dehydrogenation reactions of other compounds, the
enzyme assay was carried out using borneol as a sub-
strate based on the fact that trace amounts of borneol
and high content of camphor are found in Z. zerum-
bet. As shown in Fig. 5A, borneol was efficiently con-
verted to camphor by ZSD1, confirming that ZSD1
may have a broad substrate specificity. In this regard,
ZSD1 is similar to Adh2, a recently characterized
monoterpenoid alcohol dehydrogenase from A. annua
that exhibits dehydrogenase activity with several
monoterpene alcohol substrates including borneol [23].
(
AAK38665), Artemisia annua alcohol dehydrogenase (ADK56099),
Zea mays sex determination protein tasselseed-2 (ACG37730),
Zingiber zerumbet ZSD1 (AB480831), Arabidopsis thaliana xanthox-
in dehydrogenase (NP_175644), Citrus sinensis short-chain alcohol
dehydrogenase (ADH82118), Solanum tuberosum short-chain dehy-
drogenase ⁄ reductase (AAT75153), Citrobacter braakii (S)-6b-hy-
droxycineole dehydrogenase (GQ849481), Rhodobacter sphaeroides
galactitol dehydrogenase (ACM89305), Aedes aegypti farnesol
dehydrogenase (GQ344797), Mentha · piperita menthol dehydroge-
nase (AAQ55960), Artemisia annua broad substrate reductase ⁄
dehydrogenase (RED1) (GU167953), Digitalis lanata 3b-hydroxy-
steroid dehydrogenase (CAC936678), and Mentha · piperita (–)-iso-
piperitenol dehydrogenase (AAU20370).
Kinetic comparisons of ZSD1 for different
substrates
Because ZSD1 can act on both 8-HAH and borneol,
we conducted a kinetic analysis to evaluate the cata-
lytic preferences of ZSD1 for these two different sub-
strates. As shown in Table 1, the recombinant ZSD1
ZSD1 catalyzes 8-HAH to zerumbone in vitro
To examine whether ZSD1 could catalyze the conver-
sion of 8-HAH to zerumbone, the full-length ZSD1
was expressed in Escherichia coli BL21 (DE3) and the
recombinant protein was purified by Ni-affinity chro-
matography. Then, the purified protein was incubated
+
+
selectively uses NAD but not NADP as a cofactor
+
and the Km value for NAD is 27.3 lm with a kcat
value of 3.8 s) at pH 8.0. While the K and kcat val-
1
m
)
1
ues for borneol are 22.8 lm and 4.1 s , respectively,
the kcat ⁄K value for 8-HAH is lower than for bor-
+
+
m
with 8-HAH in the presence of NAD or NADP ,
and the product was analyzed by GC-MS. As shown
in Fig. 3, a single product peak matching the authentic
neol. Thus, it is likely that ZSD1 has a substrate pref-
erence for borneol over 8-HAH. Nevertheless, the
relatively lower catalytic efficiency of ZSD1 for that of
+
standard of zerumbone was generated when NAD
was used as cofactor, whereas no product was detected
+
in the presence of NADP (data not shown). There-
8
-HAH in vitro may be at least partially due to the
poorer solubility of 8-HAH in the aqueous reaction
solution. The high content of zerumbone and trace
amounts of 8-HAH in rhizomes of Z. zerumbet suggest
that the rate of conversion is sufficient in vivo.
fore, ZSD1 is a NAD-dependent dehydrogenase capa-
ble of oxidizing the C8 hydroxyl group of 8-HAH to
form zerumbone.
2
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S. Okamoto et al.
An SDR involved in terpene metabolism
Fig. 3. GC-MS analysis of products gener-
ated by recombinant ZSD1 with 8-HAH as
substrate. (A) Chromatogram of products
formed by incubation of 8-HAH with pro-
teins from E. coli cells carrying the empty
expression vector (pET101). Peak 1, 8-HAH.
(B) Chromatogram of products generated by
incubation of 8-HAH with recombinant
ZSD1. Peak 2, zerumbone. (C) Mass spec-
trum of peak 2. (D) Mass spectrum of
authentic zerumbone.
However, we cannot rule out the possibility that a
dehydrogenase distinct from ZSD1 can catalyze zerum-
bone formation in Z. zerumbet.
fold dinucleotide cofactor-binding motif, a Tyr-based
catalytic center with adjacent Ser and Lys residues, and
a substrate-binding site that is mainly surrounded by
hydrophobic residues (Fig. 6A). Superimposition of
ZSD1 on GatDH reveals that the two structures are
nearly identical in the N-terminal segments, despite
their low sequence identity (30%). However, in the
highly variable C-terminal region, a much larger and
flexible substrate-binding pocket was observed in
ZSD1, which may allow it to accommodate multiple
substrates, thereby conferring its versatile functional
property (Fig. 6B).
A 3D structure model of ZSD1 reveals a large,
flexible substrate-binding pocket
The bisubstrate property of ZSD1 led us to perform
molecular modeling studies on it to obtain structural
insights. A 3D structural model of ZSD1 was con-
structed by homology modeling based on the crystal
structure of Podophyllum peltatum (–)-secoisolariciresi-
nol dehydrogenase [29] (PDB code 2BGK) and super-
imposed onto Rhodobacter sphaeroides galactitol
dehydrogenase (GatDH) [30] (PDB code 3LQF), whose
Ser142 completes the Ser-Tyr-Lys triad
responsible for the catalysis of ZSD1
+
co-crystal structure with NAD and an alcohol sub-
strate is known (Fig. 6). Common to most alcohol
dehydrogenases, ZSD1 displays a typical Rossmann-
It is well known that many SDR family members
utilize a highly conserved Ser-Tyr-Lys triad (Ser and
Fig. 4. Tissue-specific expression profiles of ZSD1, ZSS1 and CYP71BA1. Quantitative RT-PCR analysis of ZSD1 (A), ZSS1 (B) and
CYP71BA1 (C) transcript levels in leaves (oblique line), stems (checked) and rhizomes (striped). Ubiquitin was used as a reference gene.
FEBS Journal 278 (2011) 2892–2900 ª 2011 The Authors Journal compilation ª 2011 FEBS
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An SDR involved in terpene metabolism
S. Okamoto et al.
Fig. 5. GC-MS analysis of products in the
enzyme assay using borneol. (A) Chromato-
gram of products formed by incubation of
borneol with recombinant ZSD1. Peak 1,
borneol; peak 2, camphor. (B) Mass spec-
trum of peak 2. (C) Mass spectrum of
authentic camphor.
Table 1. Kinetic parameters for recombinant wild-type ZSD1 and four mutants. ND, enzymatic activity was not detected.
+
NAD
8-HAH
(lM)
Borneol
(lM)
)
1
(lM)1Æs)1
)1
(lM)1Æs)1
)1
(lM)1Æs)1
)1
K
m
(lM)
k
cat (s
)
kcat ⁄ K
m
)
K
m
k
cat (s
)
k
cat ⁄ K
m
)
K
m
k
cat (s
)
k
cat ⁄ K
m
)
)
1
1
)2
)3
Wild-type
Y155A
K159A
S142A
S144A
27.3
43.7
3.8
0.1
ND
0.1
4.4
1.39 · 10
2.0 · 10
ND
58.5
78.2
ND
1.3
0.1
ND
0.1
2.9
2.2 · 10
1.0 · 10
ND
22.8
55.1
4.1
0.1
ND
0.1
4.5
1.79 · 10
2 · 10
ND
)
3
)3
ND
121
16.1
ND
111
13.2
)
4
)4
)4
8.0 · 10
138
7.0 · 10
9 · 10
3.41 · 10
)
)1
)1
2.62 · 10
28.1
1.03 · 10
YXXXK motif) to perform the oxidoreductase enzyme
reaction, with Tyr residue acting as the catalytic base
racy of our homology model. The unexpected finding
that the mutation of Ser144 enhances the catalytic effi-
ciency of ZSD1 provides a possibile way to improve
the yield of products such as zerumbone by mutagene-
sis, although further studies are required to understand
why the S144A mutant results in improved enzyme
efficiency.
[
31]. Since sequence analysis of ZSD1 has revealed a
similar motif (155Y-X-X-X-K159) (Fig. 2), we assume
that Tyr155 and Lys159 residues are predicted to be
the possible catalytic residues as reported previously.
Site-directed mutagenesis and steady-state kinetic anal-
ysis showed that the kcat ⁄ Km value of Y155A mutant
enzyme for the three substrates decreased to approxi-
mately 1⁄ 20 to 1⁄ 50 of the wild-type ZSD1, while
K159A mutant exhibited no ZSD1 activity at all
In conclusion, we cloned and characterized a bisub-
strate SDR (ZSD1) from Z. zerumbet, which acts on
both sesquiterpene alcohol 8-HAH and monoterpene
alcohol borneol. Its broad expression pattern and its
proposed flexible substrate-binding pocket are consis-
tent with its bisubstrate property.
(Table 1) [32]. These observations confirmed the key
roles for Tyr155 and Lys159 in the catalytic function
of ZSD1.
Because our homology model of ZSD1 shows that
Ser142 and Ser144 are localized in close proximity to
Tyr155 (Fig. 6A), we also created the mutants S142A
and S144A by PCR-based mutagenesis in order to
identify the third member of the catalytic triad. Kinetic
analysis indicated that the catalytic efficiency (kcat ⁄ Km)
of the S142A mutant for all three substrates was
remarkably reduced (1/200-1/20 fold) compared with
the wild-type ZSD1. In contrast, the kcat ⁄Km values of
the S144A mutant increased 2–5 fold (Table 1). These
results strongly suggest that Ser142 completes the cata-
lytic triad (Ser142-Tyr155-Lys159) responsible for
ZSD1 activity for 8-HAH and borneol. Since ZSD1
has 16 Ser residues, this result also validates the accu-
Experimental procedures
Plant materials and chemicals
Samplings of ginger plants (Zingiber zerumbet Smith) pro-
vided by Sakata Co. (Kochi, Japan) were grown in a green-
house under natural light and environmental conditions.
Mid-summer plants were used for the cDNA cloning. Rhi-
zomes, stems and leaves for analysis of the gene expression
were harvested in different seasons, immediately frozen in
liquid N , and stored at )80 ꢀC for RNA isolation. 8-HAH
2
was obtained from the Nard Institute Ltd (Hyogo, Japan).
a-humulene and other reagents were purchased from Sigma
and Aldrich Chemical Co (St. Louis, CA, USA).
2
896
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S. Okamoto et al.
An SDR involved in terpene metabolism
A
NAD+
NAD+
Lys159
Ser142
Lys159
Ser142
8-HAH
8-HAH
Tyr155
Tyr155
Ser144
Ser144
B
GatDH
ZSD1
NAD+
ZSD1
C-terminus
8-HAH
GatDH C-terminus
Fig. 6. (A) 3D structure model of monomeric ZSD1 in stereo. The NAD+ and 8-HAH structures are included. The catalytic Tyr155 (red) and
Lys159 (blue) residues as well as the potentially catalytic Ser142 (magenta) and Ser144 (yellow) residues are shown. (B) 3D structure model
of monomeric ZSD1 with NAD and 8-HAH is superimposed on monomeric GatDH (PDB code 3LQF). Green ribbon model indicates ZSD and
blue ribbon model indicates GatDH 3D structure.
Kit (Takara Bio, Ohtsu, Japan) following the manufac-
Isolation of ZSD1 cDNA
turer’s protocol. The following two gene-specific primers
Total RNA from rhizomes for RT-PCR was isolated with a
Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis,
CA, USA). Three micrograms of RNA were reverse tran-
scribed into cDNA in a 20-lL reaction with poly(dT) prim-
ing using a SuperScript III First-Strand Synthesis Kit
were used for RACE: 5¢-CGGATGTCAATGACCTTG
TCACCTGT-3¢ for 5¢RACE and 5¢-GATGAGCTTGGTC
AGCAAGTAAGCCAAC-3¢ for 3¢RACE. The PCR prod-
ucts were cloned into pGEM-T Easy Vector (Promega) and
analyzed to determine the nucleotide sequence of the ORF
in the cDNA. The cDNA fragment containing the entire
ORF from the initial codon (ATG) to the terminal codon
(TGA) for ZSD1 was then amplified by PCR using a prime
star HS DNA polymerase (Takara Bio, Ohtsu, Japan) with
a pair of oligonucleotide primers (5¢-ATGAGGTTAGA
AGGGAAAGTTGC-3¢ and 5¢-TTCGAACACTTGGAGT
GTATGG-3¢) according to the manufacturer’s instructions.
The deduced amino acid sequences of selected SDRs
were aligned using the neighbor-joining algorithm with
clustalw (http://align.genome.jp/). The phylogenetic tree
was visualized using treeview software.
(Invitrogen). Two degenerate primers, 5¢-GGIAARGTI
GCCHTIRTVACIGG-3¢ (forward) and 5¢-GGRCTNAC
RCARTTIACIC-3¢ (reverse), were used for RT-PCR. The
PCR conditions were adjusted as follows: denaturation at
94 ꢀC for 2 min, followed by 35 cycles of 94 ꢀC for 30 s,
51 ꢀC for 30 s, 72 ꢀC for 30 s, and a final extension at
72 ꢀC for 2 min. The PCR products were purified and
cloned into a pGEM-T Easy Vector (Promega, Madison,
WI, USA) and introduced into E. coli DH5a cells. The
cDNA clones were sequenced using BigDye Terminator
version 3.1 (Applied Biosystems, Foster City, CA, USA)
and an ABI3100 sequencer (Applied Biosystems). The
molecular cloning experiments in this study were mainly
performed based on Sambrook et al. (1989, Molecular clon-
ing: a laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York.). To isolate full-length
cDNA, the partial sequence was extended toward the
Bacterial expression and enzyme assay
The amplified full-length product was cloned into
a
pET101 ⁄ DTOPO vector (Invitrogen) to express ZSD1 as a
His-tagged protein. The recombinant plasmid pET-ZSD1
was transformed into E. coli TOP10F cells for sequence
5
¢- and 3¢-end with the Smart RACE cDNA Amplification
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An SDR involved in terpene metabolism
S. Okamoto et al.
+
tion of substrates or NAD systematically, while maintain-
ing the other reactant at saturation. The reaction was
characterization and into E. coli BL21 (DE3) for expres-
sion. For functional expression, recombinant E. coli cells
were grown to OD600 = 0.5–0.6 at 37 ꢀC and Luria broth
monitored at 37 ꢀC by absorbance at 340 nm. K
m
(Micha-
)
1
medium containing ampicillin (100 lgÆmL ). Cultures were
then induced by adding isopropyl-b-D-thiogalactopyrano-
side to a final concentration of 1 mm and grown for
another 3.5 h at 37 ꢀC. The cells were collected by centrifu-
gation and stored at )80 ꢀC until used. The frozen cells
were suspended in a chilled lysis buffer (50 mm Tris ⁄ HCl
elis–Menten constant) and Vmax (maximal reaction velocity)
were calculated from at least eight measurements by linear
regression from double-reciprocal plots. The kcat values
(s) ) were calculated from the Vmax values and represent
the maximal turnover rate. pH optimum was measured
using the same buffer system over a pH range of 7.0–8.0 at
0.5 pH intervals.
1
(pH 8.0), 100 mm NaCl) containing 1 mm phenylmethylsul-
phonyl fluoride and disrupted with a sonicator (Branson
advanced digital sonifier 250DA). The lysates were cleared
by centrifugation and filtered through a 0.45-lm filter. The
Transcript expression analysis
¨
filtrate was loaded onto a HisTrap HP column (AKTA
Possible traces of DNA were removed using Turbo DNA-
freeꢁ kit (Ambion, Austin, TX, USA). Then, 1 lg of RNA
was reverse transcribed into cDNA using Superscript III
first-strand synthesis Supermix for qRT-PCR (Life Technol-
ogies, Carlsbad, CA, USA). Quantitative PCR was per-
formed using a StepOnePlusꢁ real-time PCR system and a
prime system; GE Healthcare Bio-Science, Piscataway, NJ,
USA) equilibrated with the lysis buffer, and the His6
protein was eluted using an imidazole gradient from 0 to
5
00 mm. The protein eluate was further desalted into an
assay buffer (20 mm KH PO , pH 7.0, 7.5 and 8.0) by pas-
2
4
ꢂ
sage through an Econopac column (Bio-Rad, Hercules,
CA, USA), and the resulting enzyme eluate was used for
the enzyme assay. Each assay was done in a volume of
power SYBR Green Master Mix (Life Technologies,
Carlsbad, CA, USA) according to the manufacturer’s
instructions. The quantitative PCR reaction consisted of
10 lL of the master mix (Life Technologies, Carlsbad, CA,
USA), 1 lL of primers (0.4 lm) and 1 lL of cDNA in a
final volume of 20 lL. The specific primer pairs used
for amplification of the ZSD1, ZSS1 and CYP71BA1
transcripts were 5¢-GCAAGTGATGGTGGAGCAAAA-3¢
1
1
mL with 20 mm KH
2 4
PO , pH 7.0, 865 lL of enzyme,
mm NAD, 10 mm EDTA and 0.1 mm 8-HAH, borneol
or pregnenolone. After overnight incubation at 37 ꢀC, the
mixture was extracted with pentane (3 · 1 mL) and concen-
trated to a minimum volume for GC-MS analysis. Gel per-
meation chromatography of the recombinant ZSD1 was
conducted on a calibrated Sephadex 200 (GE Healthcare,
(forward), 5¢-CCGGCTACTTGTGTTGGACGT-3¢ (reverse),
5¢-GCAAGTGATGGTGGAGCAAAA-3¢ (forward), 5¢-CC
GAGCTACTTGTGTTGGACGT-3¢ (reverse), 5¢-AGCGTGC
ATAAGCAACAAGTAC-3¢ (forward) and 5¢-TGAGTT
CGGGCGAGTTGGAG-3¢ (reverse), respectively. Amplifi-
cation of the endogenous reference gene (ubiquitin) was
carried out using the following primers: 5¢-AAGGA
GTGCCCCAACGCCGAGTG-3¢ and 5¢-GCCTTCTGGT
TGTAGACGTAGGTGAG-3¢. Each sample was analyzed
in triplicate, and the results represent the normalized mean
values and sd.
Amersham Place, UK) column with 50 mm KH
2
PO
4
, pH
8
.0, containing 150 mm NaCl as running buffer at a flow
)1
rate of 0.5 mLÆmin
.
Product identification
GC-MS analysis of terpenoids was performed on a Shima-
dzu QP5050A GC ⁄ MS system with a DB-WAX column
0.25 mm internal diameter · 0.25 lm film thickness ·
(
3
0 m; Agilent Technologies, Santa Clara, CA, USA). Split
injections (1 · l) were made at a ratio of 22 : 1 with an injec-
tor temperature of 250 ꢀC. The instrument was programmed
from an initial temperature of 40 ꢀC (held for 3 min) and
Molecular modeling of ZSD1
The 3D structures of the protein complexed with the
ligands were generated with Molecular Operating Environ-
ment (moe), version 2009.10 (Chemical Computing Group,
Montreal, Canada) using the crystal structure of P. pelta-
tum ())-secoisolariciresinol dehydrogenase [29] (Protein
Data Bank code 2BGK) as a template. A ligand-free struc-
ture was generated by minimizing structural energy in the
Amber99 force field with the default parameters. For the
in silico docking simulation, the atomic coordinate of
8-HAH was prepared with the moe by energy-minimizing
the drawn structures under MMFF94x forcefield [33,34].
The simulation to dock 8-HAH to the ZSD1 model
structure was performed with moe-asedock 2005 [35]. The
ZSD1-8HAH complex model obtained was superimposed
)1
)1
increased at 3 ꢀCÆmin until 80 ꢀC, 5 ꢀCÆmin until 180 ꢀC,
)1
and 10 ꢀCÆmin until 240 ꢀC (held for 5 min). Helium was
)1
used at a constant flow of 1.8 mLÆmin . Mass spectra were
measured with a mass range m ⁄ z of 40–400, an electron volt-
age of 70 eV and an interface temperature of 230 ꢀC.
Enzyme characterization
Michaelis–Menten kinetic parameters for 8-HAH and bor-
neol were determined using purified wild-type enzymes and
four mutants. A kinetics assay was done in a volume of
1
1
mL with 20 mm KH
2 4
PO (pH 8.0), 200 lL of enzyme,
mm NAD and 10 mm EDTA by changing the concentra-
2
898
FEBS Journal 278 (2011) 2892–2900 ª 2011 The Authors Journal compilation ª 2011 FEBS

S. Okamoto et al.
An SDR involved in terpene metabolism
with the GatDH-erythritol co-crystal structure using the
4 Chene-Ming J, Vera R & Chalchat JC (2003) Chemical
composition of the essential oil from rhizomes, leaves
and flowers of Zingiber zerumbet Smith from Reunion
Island. J Essent Oil Res 15, 202–205.
moe superpose program. The figures for the protein struc-
¨
ture were generated with program pymol (Schrodinger,
New York, NY, USA).
5
6
7
8
Bhuiyan MNI, Chowdhury JU & Begum J (2009)
Chemical investigation of the leaf and rhizome essential
oils of Zingiber zerumbet (L.) Smith from Bangladesh.
Bangl J Pharmacol 4, 9–12.
Generation of ZSD1 mutants
The following primer pairs were used to introduce point
mutations into the ZSD1 ORF: Y155A forward, 5¢-GC
TGGTCCACATGGAGCAACGGGGGC AAAACATG-3¢,
and Y155A reverse, 5¢-CATGTTTTGCCCCCGTTGCTC
CATGTGGACCAGC-3¢; K159A forward, 5¢-GATAC
ACGGGGGCAGCACATGCTGTAGTAG-3¢, and K159A
reverse, 5¢-CTACTACAGCATGTGCTGCCCCCGTGTAT
C-3¢; S142A forward, 5¢-CTATAGTCTCCCTGGCCGCA
GTATCTTCTGTGATTG-3¢, and S142A reverse, 5¢-CAAT
CACAGAAGATACTGCGGCCAGGGAGACTATAG-3¢;
S144A forward, 5¢-CCTGGCCAGTGTAGCTTCTGTGAT
TGC-3¢, and S144A reverse, 5¢-GCAATCACAGAAGCTA
CACTGGCCAGG-3¢. These primers were used with the
pET-ZSD1 plasmid and the proofreading Pfu-turbo DNA
polymerase (Agilent Technologies, Santa Clara, CA, USA)
for PCR (94 ꢀC for 1 min, followed by 18 cycles of 94 ꢀC
for 30 s, 55 ꢀC for 1 min, 68 ꢀC for 5 min). Subsequently,
the PCR products were digested with DpnI (Toyobo,
Osaka, Japan) for 1 h at 37 ꢀC, and 5 lL was used to
transform E. coli strain JM109. The plasmids obtained
from the transformants were purified and sequenced.
Ozaki Y, Kawahara N & Harada M (1991) Anti-inflam-
matory effect of Zingiber cassumunar Roxb. and its
active principles. Chem Pharm Bull (Tokyo) 39,
2
353–2356.
Kirana C, McIntosh GH, Record IR & Jones GP
2003) Antitumor activity of extract of Zingiber aromat-
(
icum and its bioactive sesquiterpenoid zerumbone. Nutr
Cancer 45, 218–225.
Murakami A, Takahashi D, Kinoshita T, Koshimizu K,
Kim HW, Yoshihiro A, Nakamura Y, Jiwajinda S,
Terao J & Ohigashi H (2002) Zerumbone, a Southeast
Asian ginger sesquiterpene, markedly suppresses free
radical generation, proinflammatory protein production,
and cancer cell proliferation accompanied by apoptosis:
the alpha,beta-unsaturated carbonyl group is a prere-
quisite. Carcinogenesis 23, 795–802.
9
Murakami A, Tanaka T, Lee JY, Surh YJ, Kim HW,
Kawabata K, Nakamura Y, Jiwajinda S & Ohigashi H
(2004) Zerumbone, a sesquiterpene in subtropical
ginger, suppresses skin tumor initiation and promotion
stages in ICR mice. Int J Cancer 110, 481–490.
1
1
0 Sakinah SA, Handayani ST & Hawariah LP (2007)
Zerumbone induced apoptosis in liver cancer
cells via modulation of Bax ⁄ Bcl-2 ratio. Cancer Cell
Int 7, 4.
Acknowledgements
We thank Mr T. Ishida, Ms R. Sawa and Mr H. Miy-
awaki for providing the ginger plants. We also thank
Professor Kazutoshi Shindo and Professor Morio
Asaoka for their helpful advice. This work was
supported in part by the Research and Development
Program for New Bio-industry Initiatives (2006–2010)
from the Bio-oriented Technology Research Advance-
ment Institution (BRAIN).
1 Xian M, Ito K, Nakazato T, Shimizu T, Chen CK,
Yamato K, Murakami A, Ohigashi H, Ikeda Y &
Kizaki M (2007) Zerumbone, a bioactive sesquiterpene,
induces G2 ⁄ M cell cycle arrest and apoptosis in
leukemia cells via a Fas- and mitochondria-mediated
pathway. Cancer Sci 98, 118–126.
12 Croteau RB, Davis EM, Ringer KL & Wildung MR
2005) (-)-Menthol biosynthesis and molecular genetics.
(
Naturwissenschaften 92, 562–577.
References
1
3 Gershenzon J, Maffei M & Croteau R (1989) Biochemi-
cal and histochemical localization of monoterpene bio-
synthesis in the glandular trichomes of spearmint
1
Lechat-Vahirua I, Francois P, Menut C, Lamaty G &
Bessiere MJ (1993) Aromatic plants of French Polyne-
sia. I. Constituents of the essential oils of rhizomes of
three zingiberaceae. Zingiber zerumbet Smith, Hedychi-
um coronarium Koenig and Etlingera cevuga Smith.
J Essent Oil Res 5, 55–59.
(Mentha spicata). Plant Physiol 89, 1351–1357.
1
4 Yu F, Okamto S, Nakasone K, Adachi K, Matsuda S,
Harada H, Misawa N & Utsumi R (2008) Molecular
cloning and functional characterization of a-humulene
synthase, a possible key enzyme of zerumbone biosyn-
thesis in shampoo ginger (Zingiber zerumbet Smith).
Planta 227, 1291–1299.
2
3
Dung NX, Chinh TD, Rang DD & Leclercq PA (1993)
The constituents of the rhizome oil of Zingiber zerumbet
(L.) Sm. from Vietnam. J Essent Oil Res 5, 553–555.
1
5 Yu F, Okamoto S, Harada H, Yamasaki K, Misawa N
Srivastava AK, Srivastava SK & Shah NC (2000)
Essential oil composition of Zingiber zerumbet (L.) Sm.
from India. J Essent Oil Res 12, 595–597.
&
Utsumi R (2011) Zingiber zerumbet CYP71BA1 cata-
lyzes the conversion of alpha-humulene to 8-hydroxy-
FEBS Journal 278 (2011) 2892–2900 ª 2011 The Authors Journal compilation ª 2011 FEBS
2899

An SDR involved in terpene metabolism
S. Okamoto et al.
alpha-humulene in zerumbone biosynthesis. Cell Mol
Life Sci 68, 1033–1040.
28 Mayoral JG, Nouzova M, Navare A & Noriega FG
(2009) NADP+-dependent farnesol dehydrogenase, a
corpora allata enzyme involved in juvenile hormone
synthesis. Proc Natl Acad Sci USA 106, 21091–21096.
29 Youn B, Moinuddin SG, Davin LB, Lewis NG & Kang
C (2005) Crystal structures of apo-form and binary ⁄
ternary complexes of Podophyllum secoisolariciresinol
dehydrogenase, an enzyme involved in formation of
health-protecting and plant defense lignans. J Biol
Chem 280, 12917–12926.
1
6 Jornvall H, Persson B, Krook M, Atrian S, Gonzalez-
Duarte R, Jeffery J & Ghosh D (1995) Short-chain
dehydrogenases ⁄ reductases (SDR). Biochemistry 34,
6
003–6013.
7 Kallberg Y, Oppermann U, Jornvall H & Persson B
2002) Short-chain dehydrogenases ⁄ reductases (SDRs).
1
(
Eur J Biochem 269, 4409–4417.
1
8 de Kraker JW, Franssen MC, Dalm MC, de Groot A
&
Bouwmeester HJ (2001) Biosynthesis of germacrene
30 Carius Y, Christian H, Faust A, Zander U, Klink BU,
Kornberger P, Kohring GW, Giffhorn F & Scheidig AJ
(2010) Structural insight into substrate differentiation of
the sugar-metabolizing enzyme galactitol dehydrogenase
from Rhodobacter sphaeroides D. J Biol Chem 285,
20006–20014.
A carboxylic acid in chicory roots. Demonstration of a
cytochrome P450 (+)-germacrene a hydroxylase and
NADP+-dependent sesquiterpenoid dehydrogenase(s)
involved in sesquiterpene lactone biosynthesis. Plant
Physiol 125, 1930–1940.
1
2
2
2
2
2
9 Brosche M & Strid A (1999) Cloning, expression, and
molecular characterization of a small pea gene family
regulated by low levels of ultraviolet B radiation and
other stresses. Plant Physiol 121, 479–487.
31 Oppermann UC, Filling C, Berndt KD, Persson B,
Benach J, Ladenstein R & Jornvall H (1997) Active site
directed mutagenesis of 3 beta ⁄ 17 beta-hydroxysteroid
dehydrogenase establishes differential effects on short-
chain dehydrogenase ⁄ reductase reactions. Biochemistry
36, 34–40.
0 Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C
&
Luckner M (1999) Delta(5)-3beta-hydroxysteroid
dehydrogenase from Digitalis lanata Ehrh. – a multifunc-
tional enzyme in steroid metabolism? Planta 209, 478–486.
1 Davis EM, Ringer KL, McConkey ME & Croteau R
32 Rosano C, Bisso A, Izzo G, Tonetti M, Sturla L, De
Flora A & Bolognesi M (2000) Probing the catalytic
mechanism of GDP-4-keto-6-deoxy-d-mannose epimerase ⁄
reductase by kinetic and crystallographic characterization
of site-specific mutants. J Mol Biol 303, 77–91.
33 Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C,
Alagona G, Profeta S & Weiner P (1984) A new force
field for molecular mechanical simulation of nucleic
acids and proteins. J Am Chem Soc 106, 765–784.
34 Halgren TA (1999) MMFF VII. Characterization of
MMFF94, MMFF94s, and other widely available force
fields for conformational energies and for intermolecu-
lar-interaction energies and geometries. J Comput Chem
20, 730–748.
(2005) Monoterpene metabolism. cloning, expression,
and characterization of menthone reductases from
peppermint. Plant Physiol 137, 873–881.
2 Ringer KL, Davis EM & Croteau R (2005) Monoterpene
metabolism. Cloning, expression, and characterization of
(–)-isopiperitenol ⁄ (–)-carveol dehydrogenase of pepper-
mint and spearmint. Plant Physiology 137, 863–872.
3 Polichuk DR, Zhang Y, Reed DW, Schmidt JF & Cov-
ello PS (2010) A glandular trichome-specific monoter-
pene alcohol dehydrogenase from Artemisia annua.
Phytochemistry 71, 1264–1269.
4 Ryden AM, Ruyter-Spira C, Litjens R, Takahashi S,
Quax W, Osada H, Bouwmeester H & Kayser O (2010)
Molecular cloning and characterization of a broad sub-
strate terpenoid oxidoreductase from Artemisia annua.
Plant Cell Physiol 51, 1219–1228.
35 Goto J, Kataoka R, Muta H & Hirayama N (2008)
ASEDock-docking based on alpha spheres and excluded
volumes. J Chem Inf Model 48, 583–590.
Supporting information
2
5 Cankar K, van Houwelingen A, Bosch D, Sonke T,
Bouwmeester H & Beekwilder J (2011) A chicory
cytochrome P450 mono-oxygenase CYP71AV8 for the
oxidation of (+)-valencene. FEBS Lett 585, 178–182.
6 Takahashi S, Yeo YS, Zhao Y, O’Maille PE, Greenha-
gen BT, Noel JP, Coates RM & Chappell J (2007)
Functional characterization of premnaspirodiene
oxygenase, a cytochrome P450 catalyzing regio- and
stereo-specific hydroxylations of diverse sesquiterpene
substrates. J Biol Chem 282, 31744–31754.
The following supplementary material is available:
Fig. S1. Comparison of deduced amino acid sequence
of ZSD1 with related functionally characterized SDRs.
This supplementary material can be found in the
online version of this article.
2
Please note: As a service to our authors and readers,
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should be addressed to the authors.
2
7 Choi HW, Lee BG, Kim NH, Park Y, Lim CW, Song
HK & Hwang BK (2008) A role for a menthone reduc-
tase in resistance against microbial pathogens in plants.
Plant Physiol 148, 383–401.
2
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FEBS Journal 278 (2011) 2892–2900 ª 2011 The Authors Journal compilation ª 2011 FEBS