A novel reductase participating in the hydrogenation of an exocyclic C-C double bond of enones from Nicotiana tabacum

Article abstract of DOI:10.1246/bcsj.75.813

A novel 74 kDa enone reductase which catalyzes the reduction of the exocyclic C-C double bond of enones was isolated from cultured cells of Nicotiana tabacum. The reductase was found to catalyze the enantiofacially selective reduction of the C-C double bond of 2-alkylidenecyclohexanone to give optically active (S)-2-alkylcyclohexanone.

Full text of DOI:10.1246/bcsj.75.813

c. Jpn.
© 2002 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 75, 813–816 (2002)
813
e Chemical
ciety of Ja-
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e Chemical
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n
A Novel Reductase Participating in the Hydrogenation of an Exocyclic
C–C Double Bond of Enones from Nicotiana tabacum
Enone Reductase from Nicotiana tabacum
K. Shimoda et al.
Kei Shimoda, Shunsuke Izumi, and Toshifumi Hirata*
02
3
6
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,
1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526
SJA8
09-2673
331
(Received September 17, 2001)
A novel 74 kDa enone reductase which catalyzes the reduction of the exocyclic C–C double bond of enones was
isolated from cultured cells of Nicotiana tabacum. The reductase was found to catalyze the enantiofacially selective re-
duction of the C–C double bond of 2-alkylidenecyclohexanone to give optically active (S)-2-alkylcyclohexanone.
01
01
Enone reductases occur widely in plants and microorgan-
isms. Many studies on the reduction of the C–C double bond
of enones by plant cell cultures and microorganisms have re-
vealed that there are two different types of enone reductase
with respect to the substrate specificity; one is responsible for
reducing the endocyclic double bond and the other is for re-
ducing the exocyclic double bond of enones.1–8 Recently, we
reported the isolation of two enone reductases participating in
the reduction of the endocyclic C–C double bond of enones
from cultured cells of N. tabacum; carvone reductase was able
to reduce only a C–C double bond bearing a hydrogen atom at
the β position to the carbonyl group,9 and verbenone reductase
was capable of reducing a broad range of enones.10,11 We have
3.6
4
Fig. 1. Elution profiles of enone reductases on DEAE-Toyo-
pearl column chromatography. : 74 kDa,
44 kDa, and : 90 kDa enone reductase.
now isolated a novel reductase which participates in the reduc-
tion of the exocyclic C–C double bond of enones, such as pule-
gone (1), from N. tabacum,12 and here report on the details
concerning the characterization of the reductase and the ste-
reospecificity in the enzymatic reduction of enones.
:
ed in our previous paper. However, the 74 kDa reductase was
different from the known reductases. Chromatographic purifi-
cation of the 74 kDa reductase resulted in a 160-fold enhance-
ment of the reductase activity, as shown in Table 1. SDS-
PAGE of the enone reductase revealed a single protein band
corresponding to a molecular mass of 37 kDa, as shown in Fig.
2. The molecular mass in the native state was estimated to be
74 kDa by gel filtration chromatography on a Sephadex G-150
column using marker proteins. These observations indicate
that the native enzyme is a dimeric structure of 37 kDa sub-
units. The enzyme reaction was found to require NADH or
Results and Discussion
A soluble cell-free extract was obtained from cultured cells
of Nicotiana tabacum. The crude extract was subjected to
chromatography on a DEAE-Toyopearl column to give three
reductases of 44, 74 and 90 kDa, participating in the reduction
of the C–C double bond of enones (see Fig. 1). The 90 kDa
and 44 kDa reductases were identified as the verbenone
reductase11 and the carvone reductase,9 respectively, as report-
Table 1. Purification of a 74 kDa Enone Reductase from Cultured Cells of N. tabacum
Total protein
Total activity
Specific activity
Step
Fold
mg
120
8
0.5
0.07
Ua)
12
3
1.5
1.1
U mg−1
0.1
0.4
3
16
Crude extract
1
4
30
DEAE-Toyopearl
Hydroxylapatite
Red Toyopearl
160
a) One unit of the reductase activity is defined as the quantity of enzyme that
reduces 1 µmol of (R)-Pulegone (1) per one minute under the standard assay con-
dition.

814 Bull. Chem. Soc. Jpn., 75, No. 4 (2002)
Enone Reductase from Nicotiana tabacum
Fig. 2. SDS-gel electrophoretic analyses of a 74 kDa enone
reductase. Lane 1: Crude extract; Lane 2: Red Toyopearl
Fr.
Chart 1. Formulas 1–14.
Fig. 3. Comparison of amino acid sequences of the 74 kDa
reductase (p74 red.) and the reported sequences deduced
from nucleotide sequences of four genes for isovaleryl-
CoA dehydrogenases: S. tuberosum-a from Solanum
tuberosum (Gene Bank accession no. AJ278988) [17], S.
tuberosum-b from Solanum tuberosum (AJ278987) [17],
A. thaliana-a from Arabidopsis thaliana (AAD45605)
[17], and A. thaliana-b from Arabidopsis thaliana
Table 2. Reduction of Enones, 1–10, with the p74 Reductase
from Cultured Cells of N. tabacum
Substrate
Product
Conv.a)/%
e.e.b)
> 99d)
—
Config.c)
1
2
11
—
—
—
—
12
13
—
—
14
> 99
R
—
—
—
—
S
0
0
3
—
4
0
—
5
0
—
(T47470) [14]. Numbers indicate positions relative to the
putative initial methionine or the amino terminus.
6
7
8
> 99
85
0
97
75
—
S
—
—
—
NADPH as the coenzymes, and NADPH was a better electron
donor by a factor of 3 compared with NADH. The enzyme had
a pH optimum at 7.1 in Na–Pi buffer.
9
10
0
37
—
—
The partial amino acid sequences of the enone reductase
were analyzed with an automated protein sequencer. However,
the amino terminus of the reductase was blocked. The reduc-
tase, therefore, was digested with lysylendopeptidase directly
on the gel of SDS-PAGE. The amino acid sequence for a 29
kDa peptide fragment was found to be Thr-X-Leu-Leu-Phe-
Asp-Asp-Thr-Gln-Lys-Gln-Phe-Lys. A comparison of the
peptide sequence against a protein database using the NCBI
“blastp” program showed a significant homology with isov-
aleryl-CoA dehydrogenase (Fig. 3).13–15
In order to examine the substrate specificity and stereospec-
ificity in the reduction of enones with the 74 kDa reductase,
enzymatic reductions were carried out using several enones, 1–
10, as substrates (Chart 1). When monoterpene enones, such
as (R)-pulegone (1), (R)-carvone (2), (S)-carvone (3), (1S,5S)-
verbenone (4), and (1R,5R)-verbenone (5), were used as the
substrate, the 74 kDa reductase catalyzed a stereospecific re-
duction of (R)-pulegone (1), which has the exocyclic C–C dou-
ble bond to give (1R,4R)-isomenthone (11) (> 99% d.e.), but
a) The conversions were expressed as the percentage of the
products in the enzymatic reaction mixture. b) Enantio-
meric excess on the basis of GLC on CP-cyclodextrin β
236 M-19 column. c) Configuration at the α-position to
the carbonyl group of the products. d) Diastereomeric
excess.
no reduction occurred in the case of the enones, 2–5, which
have an endocyclic C–C double bond, as shown in Table 2.
On the other hand, 2-alkylidenecyclohexanones (6 and 7),
having an exocyclic C–C double bond, were converted to the
corresponding saturated ketones (Table 2). The reduction at C-
2 of the enones took place stereoselectively from the re–re face
of the C–C double bond of 6 and the si–re face of that of 7 to
give ketones having an S configuration at C-2. However, no 2-
methylcyclohex-2-en-1-one (8) having the endocyclic C–C
double bond was suitable as a substrate for the reductase. In
the case of the reduction of acyclic enones, (E)-2-octenal (9)
was not reduced, but 1-octen-3-one (10) was reduced to give

K. Shimoda et al.
Bull. Chem. Soc. Jpn., 75, No. 4 (2002) 815
(neat); lit.2: [α]D25 −59.7° (neat)} and (S)-carvone (3) {GLC >
99%, [α]D25 +57.1° (neat); lit.2: [α]D25 +60.0 (neat)} were pur-
chased from Sigma. (1S,5S)-Verbenone (4) {99% pure on GLC,
[α]D25 −209.3° (neat); lit.11: [α]D25 −208° (neat)} and (1R,5R)-ver-
benone (5) {99% pure on GLC, [α]D25 +210.5° (neat); lit.11: [α]D25
+210° (neat)} were prepared from (−)- and (+)-α-pinenes, re-
spectively, by oxidation with t-butyl chromate.11,18
2-Methylidenecyclohexanone (6), 2-propylidenecyclohexanone
(7) and 2-methylcyclohex-2-en-1-one (8) were prepared as de-
scribed previously.12 (E)-2-Octanal (9) and 1-octan-3-one (10)
were purchased from Sigma.
octan-3-one (14), as shown in Table 2.
The Km values in the enzymatic reduction of enones 1, 6, 7
and 10, were determined to be 25, 35, 150 and 83 µM, respec-
tively. These facts suggested that the 74 kDa enone reductase
is characteristic to the hydrogenation of the exocyclic C–C
double bond of enones, especially pulegone (1).
Thus, the 74 kDa enone reductase, named pulegone reduc-
tase, was isolated from cultured cells of N. tabacum. We re-
cently reported that both the carvone reductase and verbenone
reductase from N. tabacum reduced the endocyclic C–C dou-
ble bond of enones, but no reduction occurred for enones with
an exocyclic C–C double bond.12 The substrate specificity and
the stereoselectivity of the pulegone reductase were quite dif-
ferent from those of the carvone reductase and verbenone re-
ductase. The pulegone reductase was able to reduce enantiose-
lectively the exocyclic C–C double bond of enones to afford
optically active (S)-2-alkylated ketones. The substrate speci-
ficity of the reductase was similar to that of the Reductase-Ⅱ
(132 kDa), which was isolated from N. tabacum by Tang et
al.16 However, the pulegone reductase was apparently different
from the Reductase-Ⅱ with respect to the molecular mass and
the optimum pH. On the other hand, two enone reductases cat-
alyzing the enantioselective reductions of (E)- and (Z)-2-phe-
nyl-2-butenal into (R)-2-phenylbutanal were recently charac-
terized from Saccharomyces cerevisiae, but both of them were
reported to hardly catalyze the reduction of pulegone (1).17
Therefore, the pulegone reductase isolated here is different
from the enone reductases from S. cerevisiae with respect to
the substrate specificity. It is worth noting that the asymmetric
synthesis of highly optically pure 2-alkylated ketones could be
achieved by a selective use of the enone reductases isolated
from cultured cells of N. tabacum.
Enzyme Preparation. All of the operations were carried out
at 4 °C. The cultured cells (200 g) of N. tabacum (cultivated for 3
weeks) were ground in a Waring blender with 400 mL of 0.1 M
(1 M = 1 mol dm−3) Na–Pi buffer (pH 6.8) containing 10 mM 2-
mercaptoethanol and 5 mM dithiothreithol. The resulting slurry
was filtered through three layers of cheesecloth. The filtrate was
centrifuged at 10000 g for 15 min. The supernatant was fraction-
ated stepwise by the addition of (NH4)2SO4, and the fraction ob-
tained between 40 and 60% satn was collected by centrifugation.
The pellet was dissolved in 30 mL of 50 mM Tris-HCl buffer (pH
8.0) containing 1 mM 2-mercaptoethanol and 1 mM dithiothrei-
thol (buffer A) and the crude enzyme soln was freed from
(NH4)2SO4 by passing through a Sephadex G-25 column (3×40
cm) equilibrated with buffer A. The desalted proteins were ap-
plied to a DEAE-Toyopearl column (2×20 cm) equilibrated with
buffer A. The enzymes were eluted with 200 mL of buffer A con-
taining a 0–0.5 M linear gradient of NaCl to give an enone reduc-
tase fraction, which catalyzed the reduction of the C–C double
bond of pulegone (1). The reductase fraction was further applied
to a hydroxylapatite column (1×10 cm) equilibrated with buffer
A. The column was washed with 100 mL of buffer A and the en-
zymes were eluted with 50 mL of a linear gradient of 50–500 mM
Tris-HCl buffer. The active fraction was collected and concentrat-
ed by ultrafiltration and subsequently subjected to further purifica-
tion on a Red-Toyopearl column (1×10 cm) equilibrated with
buffer A. After non-adsorbed proteins were eluted, adsorbed pro-
teins were eluted with buffer A containing a 0–1.0 M linear gradi-
ent of NaCl. The active fractions were collected and used as the
purified enzyme (pulegone reductase).
SDS-PAGE was performed by a standard protocol by
Laemmli19 on a vertical slab gel. Samples were treated for 5 min
at 100 °C in the presence of 6% SDS before application to the gel.
After electrophoresis, the protein bands were visualized by stain-
ing with Coomassie Brilliant Blue. The molecular mass of the en-
zyme was calculated by using the LMW electrophoresis calibra-
tion kit (Pharmasia). Gel filtration chromatography was carried
out on a Sephadex G-150 column (1×100 cm) with 0.1 M Na–Pi
buffer (pH 7.0) containing 0.3 M NaCl by use of the calibration
proteins with the indicated molecular mass, aldolase (158 k), bo-
vine serum albumin (67 k), ovalbumin (43 k) and ribonuclease A
(13.7 k), as references.
Experimental
General. Analytical and prep. TLC were carried out on glass
sheets (0.25 mm and 0.5 mm) coated with silica gel (Merck silica
gel 60; GF254). GLC analyses were carried out with FID and a
glass column (3 mm×2 m) packed with 15% DEGS on Chro-
mosorb W (AW-DMCS; 80–100 mesh) at 120 °C, or a capillary
column (0.25 mm×25 m) coated with 0.25 µm CP cyclodextrin β
236M-19 (WCOT) using N2 as a carrier gas (column temp: 100
°C, split ratio: 50, make up: 50 mL min−1). GC-MS (Shimadzu)
was carried out on a mass spectrometer equipped with an EI ion
source (70 eV) and a gas chromatograph equipped with a capillary
column (0.25 mm×25 m) coated with 0.25 µm OV-101. 1H NMR
spectra were obtained on a JEOL GSX-500 spectrometer using
tetramethylsilane as an internal standard in CDCl3.
Plant Cells and Substrates. Prior to their use in experi-
ments, suspended cells of N. tabacum2 were cultured on a rotary
shaker (75 rpm) at 25 °C for 3 weeks in 500 mL conical flasks
containing 200 mL Murashige and Skoog's medium under illumi-
nation (4000 lux).
The pH optimum in the reduction of pulegone (1) with the
enone reductase was determined by an enzyme reaction in a 100
mM Na–Pi buffer with the pH adjusted from 6.5 to 8.0.
Enzyme Activity Assay and Determination of Kinetic Para-
meters. The standard assay mixture (2 mL) was composed of
the enzyme prepn, 200 µmol NADPH, 100 µmol pulegone (1) and
0.2 mL 1% Triton X-100 in 50 mM Tris-HCl buffer (pH 7.0). The
mixture was incubated at 35 °C for 8 h and then extracted with
ether (3 mL×3). The organic layer was dried over Na2SO4 and the
NAD+, NADP+, NADH and NADPH were purchased from
Boehringer. FAD and FMN were from Sigma. DEAE-Toyopearl,
Red-Toyopearl and a TSK G3000 SW HPLC column were from
Tosoh Co. Ltd. Hydroxylapatite was obtained from Wako Chemi-
cal Co. Ltd.
(R)-Pulegone (1) {GLC > 99%, [α]D25 +22.3 (neat); lit.4: [α]D25
+22.4° (neat)}, (R)-carvone (2) {99% pure on GLC, [α]D25 −60.1°

816 Bull. Chem. Soc. Jpn., 75, No. 4 (2002)
Enone Reductase from Nicotiana tabacum
solvent was removed by evapn. The concentrated ether layer was
subjected to GLC and GC-MS analyses. One unit of activity is
defined as the enzyme amount of reducing 1 µmol of the enone
per min. The protein concentration was determined according to
the Bradford method with BSA as a standard.20
and GC-MS spectra.
References
1
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ity was measured using pure enzymes under standard assay condi-
tions. The initial rates of substrate reduction were obtained for
each substrate concentration, varying between 20 and 500 µM
while maintaining the concentration of NADPH at 3 mM; also, the
apparent Michaelis constants (Km) were calculated from direct lin-
ear plots.
Amino Acid Sequence of the Enzyme. The purified en-
zyme was subjected to SDS-PAGE (12.5% gel), and then blotted
onto a poly(vinylidene disulfide) membrane (Immobilon PSQ, Mil-
lipore) by semi-dry blotting method. Attempts to sequence the re-
ductase using Edman degradation with an Applied Biosystems
Model 473A pulsed liquid sequencer with an online phenylthiohy-
dantoin amino acids analyzer were unsuccessful. The reductase,
therefore, was digested with lysyl endopeptidase directly in the
SDS-gels to give two main peptid fragments (29 kDa and 20 kDa).
The resulting peptides were sequenced. The obtained amino acid
sequences were analyzed by the database screening with the NCBI
“blastp” program.
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6.5 Hz, 7-Me), 0.95 (3H, d, J = 6.5 Hz, 9-Me), 0.98 (3H, d, J =
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1
6.5 Hz, 10-Me) by direct comparison on GLC, GC-MS and H
NMR with those of the authentic sample.4 The products in the re-
duction of 2-alkylidenecyclohexanones (6 and 7) were identified
as their corresponding 2-alkylcyclohexanones (12 and 13). The
absolute configurations and enantiomeric purities of the 2-alkylcy-
clohexanones obtained were determined by the circular dichroism
(CD) spectra, and the peak area of the corresponding enantiomers
by GLC analyses on CP cyclodextrin β 236M-19 column. The
CD data of the products were as follows. 12: 97% e.e. on GLC,
[θ]288 +960 (c 0.25, MeOH) {lit.21 [θ]288 −987 for R enantiomer}
and 13 75% e.e. on GLC, [θ]288 +1860 (c 0.25, MeOH) {lit.22
[θ]288 +2480}.
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