8-Dimethylallylnaringenin 2′-hydroxylase, the crucial cytochrome P450 mono-oxygenase for lavandulylated flavanone formation in Sophora flavescens cultured cells

Article abstract of DOI:10.1016/S0031-9422(01)00270-9

8-Dimethylallylnaringenin (8-DMAN) 2′-hydroxylase, which is indispensable for the formation of a lavandulylated flavanone, sophoraflavanone G, was detected in cell suspension cultures of Sophora flavescens. The enzyme catalyzes the 2′-hydroxylation of 8-D

Full text of DOI:10.1016/S0031-9422(01)00270-9

Phytochemistry 58 (2001) 671–676
www.elsevier.com/locate/phytochem
0
8-Dimethylallylnaringenin 2 -hydroxylase, the crucial cytochrome
P450 mono-oxygenase for lavandulylated flavanone formation in
Sophora flavescens cultured cells
a
b
b,
Hirobumi Yamamoto , Atsushi Yatou , Kenichiro Inoue *
aMedicinal Plant Garden, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
bDepartment of Pharmacognosy, Gifu Pharmaceutical University, 6-1 Mitahora-higashi 5 chome, Gifu 502-8585, Japan
Received 9 March 2001; received in revised form 18 May 2001
Abstract
0
8
-Dimethylallylnaringenin (8-DMAN) 2 -hydroxylase, which is indispensable for the formation of a lavandulylated flavanone,
0
sophoraflavanone G, was detected in cell suspension cultures of Sophora flavescens. The enzyme catalyzes the 2 -hydroxylation of 8-
DMAN to leachianone G, and is tightly bound to the membrane. It required NADPH and molecular oxygen as cofactors, and was
inhibited by several cytochrome P450 inhibitors such as carbon monoxide and cytochrome c, indicating that the reaction is mediated
by a cytochrome P450 monooxygenase. The optimum pH of 8-DMAN 2 -hydroxylase was 8.5, and the enzyme hydroxylated only 8-
DMAN. Apparent Km values for 8-DMAN and NADPH of the enzyme were 55 and 34 mM, respectively. # 2001 Elsevier Science
Ltd. All rights reserved.
0
Keywords: Sophora flavescens; Leguminosae; Cell suspension cultures; Biosynthesis; Cytochrome P450 mono-oxygenase; Prenylated flavanones;
8-Dimethylallylnaringenin; Leachianone G; Sophoraflavanone G
1
. Introduction
intact plant (Yamamoto et al., 1991). We have also
found that the production of SFG in the cultured cells was
stimulated by the addition of some elicitors (Yamamoto et
al., 1995) as well as cork tissues (Yamamoto et al., 1996,
1999). Recently, we reported naringenin 8-dimethylallyl-
transferase in the microsomal fraction from S. flavescens
cultured cells that prenylated C-8 of (ꢀ)-(2S)-naringenin
to afford (ꢀ)-(2S)-8-dimethylallylnaringenin (8-DMAN;
sophoraflavanone B) as the sole prenylated product
(Yamamoto et al., 2000a). The enzyme prenylated nar-
ingenin more effectively compared to other flavanone
Prenylated flavonoids are widely distributed in legu-
minous, moraceous, berberidaceous and iridaceous
plants (Barron and Ibrahim, 1996). In most cases, one or
two dimethylallyl moieties are bound to the A or B rings
of flavonoids, and sometimes cyclization of a double bond
of this side chain with a neighboring phenolic OH
function forms a chromene ring as seen in euchresta-
flavanone c and osajin in Euchresta japonica (Shirataki et
al., 1982). Furthermore, it is characteristic of prenylated
flavonoids in Sophora flavescens, a leguminous plant, to
possess a lavandulyl group, an irregular monoterpenoid
0
derivatives. Interestingly, when 2 -hydroxynaringenin
was used as a prenyl acceptor, the formation of
SFG, a lavandulylated flavanone, together with that
0
unit, at the C-6 or C-8 position of 2 -hydroxyflavanone
(
0
Hatayama and Komatsu, 1971; Wu et al., 1985a,b,c,
986). For elucidation of the formation mechanism of this
of 2 -hydroxy-8-dimethylallylnaringenin (leachianone
0
1
G, LG) was observed. At that time, any naringenin 2 -
hydroxylation activity could not be detected in S. fla-
unique prenyl group, we have established cell suspension
cultures of S. flavescens which produced sophoraflavanone
G (SFG), one of the main lavandulylated flavanones in the
0
vescens cells. These data suggest that the 2 -hydroxy
group of flavanone plays an important role for the for-
mation of a lavandulyl group in S. flavescens cells, and
that a lavandulyl group is formed by two-step dimethy-
*
Corresponding author. Tel.: +81-58-237-3931; fax: +81-58-237-
979.
E-mail address: inoueken@gifu-pu.ac.jp (K. Inoue).
0
lallylations, between which 2 -hydroxylation is inserted
(Fig. 1).
5
0
031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00270-9

672
H. Yamamoto et al. / Phytochemistry 58 (2001) 671–676
Fig. 1. Proposed biosynthetic route to sophoraflavanone G from naringenin in Sophora flavescens cultured cells. DMAPP: dimethylallyl diphos-
phate.
In the present paper, we report the detection and
0
Table 1
Activity and cofactor requirement of 8-DMAN 2 -hydroxylase from
0
characterization of 8-DMAN 2 -hydroxylase, essential
for the formation of lavandulylated flavanones, in S.
flavescens cultured cells.
cultured cells of Sophora flavescensa
Conditions
Relative activity (%)
Complete assay
Heat-denatured enzyme
100.0
0.0
0.0
2
. Results and discussion
-
-
-
-
-
-
8-DMAN
Protein
NADPH
0.0
0.0
0
2
.1. Detection of 8-DMAN 2 -hydroxylase activity
1 mM NADPH+1 mM NADH
1 mM NADPH+1 mM FAD
1 mM NADPH+1 mM FMN
16.2
7.3
When crude cell-free extracts or the microsomal frac-
9.5
tion from S. flavescens cell suspension cultures were incu-
bated with (ꢀ)-(2S)-8-DMAN and NADPH, the
formation of a new compound whose retention time and
UV absorption pattern were completely identical to those
of leachianone G (LG) was observed in HPLC-photo-
diode array analysis. The reaction product was isolated on
a preparative scale, and was identified as LG by the direct
comparison of its EI-MS spectrum with that of authentic
LG. More than 90% of the hydroxylation activity still
remained in the twice-washed 100,000ꢁg pellets, indi-
cating that the hydroxylase was tightly bound to the
membrane fraction of the cells (data not shown). The
activity depended on the presence of the active enzyme,
a
The complete assay contained microsomal protein (430 mg), 1
mM (S)-8-DMAN, and 1 mM NADPH. Incubations were carried out
as described in Experimental (incubation time: 120 min). 8-DMAN
0
2
-hydroxylase activity in the complete assay was 13.3 pkat/mg
protein.
0
2.2. Biochemical properties of 8-DMAN 2 -hydroxylase
0
The pH optimum of 2 -hydroxylase activity was 8.5 in
Tris–HCl buffer. The activity in glycine-NaOH buffer at
pH 8.5 was only 60% of that observed in Tris–HCl-buf-
fer (data not shown). The hydroxylation of 8-DMAN
was linear with time up to 60 min and with protein con-
centration from 0.06 mg to 0.6 mg in the standard assay.
8-DMAN and NADPH, and the latter could not be
replaced by NADH, FAD or FMN (Table 1).
0
The substrate specificity of 8-DMAN 2 -hydroxylase for

H. Yamamoto et al. / Phytochemistry 58 (2001) 671–676
673
several flavonoids including chemically synthesized dime-
thylallylated naringenins (Tahara et al., 1994) was investi-
gated. Among these, only 8-DMAN was hydroxylated,
but other flavonoids examined, kaempferol, quercetin,
apigenin, genistein, naringenin, 6-dimethylallylnaringenin,
0
0
3
ingenin, 6 ,6 -dimethyl-4 ,5 -dihydropyrano-[2 ,3 :6,7]
-dimethylallylnaringenin,
00
3 ,6-di-(dimethylallyl)nar-
00 00
00
00 00
00
00
00 00
00
naringenin and 6 ,6 -dimethyl-4 ,5 -dihydropyrano-[2 ,
3
00
:7,8]naringenin were not hydroxylated (data not shown).
The optimal concentrations of (ꢀ)-(2S)-8-DMAN and
NADPH for enzymatic hydroxylation were between 0.5
and 1 mM, and the apparent Km values for 8-DMAN
and NADPH were calculated as 55 and 34 mM, respec-
tively (Figs. 2 and 3).
0
2.3. Involvement of cytochrome P450 in 8-DMAN 2 -
hydroxylation
0
Fig. 3. Dependence of 8-DMAN 2 -hydroxylase on the concentration
of NADPH measured with the microsomal fraction of Sophora fla-
vescens. Insert: Lineweaver–Burk plot with varying concentrations
0
In addition to NADPH, 8-DMAN 2 -hydroxylase
required molecular oxygen (Table 2). Replacement of
O in the reaction mixture by N prior to the incubation
(0.01–0.5 mM) to calculate the apparent K
m
value for NADPH.
2
2
led to a significant decrease in the activity. When O in
2
the reaction mixture was completely removed by the
addition of an excess of glucose, glucose oxidase and
catalase (Kochs and Grisebach, 1987), the formation of
LG was hardly detectable (Table 2). These findings
suggested the participation of cytochrome P450 haemo-
protein in the hydroxylation of 8-DMAN. To confirm
this hypothesis, the effects of several cytochrome P450
inhibitors were examined (Table 3). Carbon monoxide,
a well-known inhibitor of cytochrome P450 (Ortiz de
Montellano and Correia, 1995), strongly inhibited the
Table 2
Oxygen dependence of 8-DMAN 2 -hydroxylase activity
0
a
Conditions
Air bubbled
Relative activity (%)
100.0
13.7
8.8
N
2
bubbled
Glucose+glucose oxidase
catalase
+
+
+ Glucose+heat-denatured
glucose oxidase+catalase
89.4
a
Incubations were carried out as described in Experimental. 8-
0
DMAN 2 -hydroxylase activity in air bubbled control was 12.6 pkat/
mg protein
Table 3
Effects of cytochrome P450 inhibitors on 8-DMAN 2 -hydroxylase
0
activitya
Inhibitor
Relative activity (%)
Air (dark)
100.0
14.5
77.0
100.0
94.7
76.0
63.7
69.9
29.1
109.0
CO/O
CO/O
None
2
(9:1, dark)
(9:1, light)
2
Ancymidol 1 mM
Metyrapone 1 mM
.
Miconazole 1 mM
Tropolon 1 mM
Cytochrome 10 mM
KCN 1 mM
0
Fig. 2. Dependence of 8-DMAN 2 -hydroxylase on the concentration
a
of (ꢀ)-(2S)-8-dimethylallylnaringenin (DMAN) measured with the
microsomal fraction of Sophora flavescens. Insert: Lineweaver-Burk
plot with varying concentrations (0.01–1 mM) to calculate the appar-
Incubations were carried out as described in Experimental. White
0
light (20,000 lx) was used for the reversion experiment. 8-DMAN 2 -
hydroxylase activity in the control assay (Air, Dark) was 15.5 pkat/mg
protein.
ent K
m
value for (ꢀ)-(2S)-8-DMAN.

674
H. Yamamoto et al. / Phytochemistry 58 (2001) 671–676
hydroxylation in the dark, and this inhibition was par-
tially reversible by the illumination. Cytochrome c, which
competitively removes electrons from the NADPH-cyto-
chrome c reductase complex (Ortiz de Montellano and
Correia 1995), was the strongest inhibitor tested. Even at
1994; Asada et al., 2000) which is operative in the for-
mation of isoprenoids biosynthesized in the plastids
such as monoterpenoids (Eisenreich et al., 1997; Adam
et al., 1998; Turner et al., 1999), as well as b-carotene
and prenyl chains of chlorophylls (Lichtenthaler et al.,
1997; Disch et al., 1998). In contrast, in Lithospermum
erythrorhizon cultured cells, geranyl pyrophosphate
supplied from the mevalonate pathway in the endo-
plasmic reticulum/cytosol (Sommer et al., 1995; Li et al.,
1998) was utilized for the prenylation of p-hydro-
xybenzoic acid which took place in the endoplasmic reti-
1
7
0 mM cytochrome c, the activity was inhibited by about
0%. Other inhibitors, ancymidol (Graebe 1987), metyr-
apone and miconazole (Ortiz de Montellano and Correia
995) and tropolone (Rueffer and Zenk, 1994) showed less
1
0
pronounced inhibition of 8-DMAN 2 -hydroxylation
similar to their effects on other plant cytochrome P450
mono-oxygenases (Tang and Suga 1994; Petersen, 1997;
Yamamoto et al., 2000b). Potassium cyanide, an inhi-
bitor of peroxidase, hardly affected the activity (Saun-
0
culum (Yamaga et al., 1993). In SFG biosynthesis, 2 -
hydroxylation catalyzed by a cytochrome P450 mono-
oxygenase occurred between two prenylations, and most
of cytochrome P450 enzymes were associated with the
endoplasmic reticulum (von Wachenfeldt and Johnson,
1995). Thus, in Sophora cells, the reaction site of the two
prenylations in relation to the source of two dimethy-
lallyl moieties might be different. For the complete elu-
cidation of the lavandulyl moiety formation in Sophora
cells, it will be necessary to clarify the subcellular loca-
lization of the three membrane-bound enzymes includ-
ders et al., 1964). These results indicate that 8-DMAN
0
2 -hydroxylase belongs to the group of cytochrome P450
monooxygenases.
Several studies have been done on the flavonoid
hydroxlases, most of which are cytochome P450-depen-
0
0
dent. 2 - and 3 -Hydroxylases from Cicer arietinum were
0
specific solely for 4 -methoxyisoflavones but the sub-
strate specificity for flavanones was not described
0
(Clemens et al., 1993; Overkamp et al., 2000). Hetero-
logous expression in yeast demonstrated that CYP81E1,
ing 8-DMAN 2 -hydroxylase and two prenyltransferases
as well as the origin of dimethylallyl diphosphate.
a cytochrome P450 cDNA cloned from Glycyrrhiza
0
echinata, is isoflavone 2 -hydroxylase, which catalyzed
the hydroxylation of isoflavones such as daidzein and
formononetin but did not that of flavanones (Akashi et
al., 1998). Furthermore, up to the present time, nothing
3. Experimental
3.1. Chemicals
0
has been reported about flavanone 2 -hydroxylase. The
cytochrome P450-dependent hydroxylase investigated in
this paper, which catalyzes the hydroxylation of 8-
dimethylallynaringenin alone, accordingly, is the first
(ꢀ)-(2S)-8-Dimethylallylnaringenin was enzymatically
synthesized using naringenin 8-dimethylallyltransferase
from Sophora flavescens cultured cells (Yamamoto et
0
0
example of the 2 -hydroxylase of prenylated flavanones.
0
al., 2000a). 6-Dimethylallylnaringenin, 3 -dimethylallyl-
naringenin, 3 ,6-di-(dimethylallyl)naringenin, 6 ,6 -di-
0
0
0
0
0
Recently we demonstrated that the presence of 2 -OH
00 00 00 00 00
methyl-4 ,5 -dihydropyrano-[2 ,3 :6,7]naringenin and 6 ,
function of naringenin is indispensable for the forma-
tion of SFG in S. flavescens cultured cells (Yamamoto et
al., 2000a). We also speculated that a lavandulyl group at
C-8 of naringenin is formed by phased dimethylallyla-
0
0
00 00
00 00
6 -dimethyl-4 ,5 -dihydropyrano-[2 ,3 :7,8]naringenin
were chemically synthesized from naringenin and 2-
methyl-3-buten-2-ol in the presence of boron trifluoride/
etherate according to the method of Tahara et al.
(1994). Apigenin, genistein, kaempferol, naringenin and
quercetin were purchased from Funakoshi, Japan.
0
tions, between which the hydroxylation at C-2 intervenes,
0
since naringenin 2 -hydroxylase activity was hardly
detectable in Sophora cells. In this paper, we demonstrated
that 8-DMAN was converted to LG by membrane-bound
0
8
-DMAN 2 -hydroxylase from S. flavescens cultured cells
3.2. Plant material and culture method
0
and that 8-DMAN 2 -hydroxylase is a member of the
cytochrome P450 mono-oxygenase family. It follows
from these results that SFG is biosynthesized from nar-
The origin and subculturing of callus cultures
(Yamamoto et al., 1991) and the establishment of cell
suspension cultures (Yamamoto et al., 1996) of Sophora
flavescens were described in the previous papers.
ingenin via 8-DMAN and LG, successively, by naringenin
0
8
-dimethylallyltransferase, 8-DMAN 2 -hydroxylase and
an uncharacterized membrane-bound lavandulyl moiety
synthase (Yamamoto et al., 2000a) in S. flavescens cells
(
3.3. Enzyme preparation
Fig. 1).
In leguminous plants, prenylation of flavonoids is
For enzyme preparation, all procedures were carried
ꢂ
known to take place in the plastids (Biggs et al., 1990).
Furthermore, their dimethylallyl groups are found to be
derived from the non-mevalonate pathway (Hano et al.,
out at 4 C. Thirty grams of S. flavescens cells cultured for
8–10 days were homogenized by a Teflon homogenizer in
100 mM K-Pi buffer (pH 6.5, 60 ml) containing 10 mM

H. Yamamoto et al. / Phytochemistry 58 (2001) 671–676
675
DTT and 3 g PVPP. The homogenate was centrifuged at
1
3.7. Isolation and identification of the reaction product
2,000ꢁg for 20 min, and the supernatant was further
ultracentrifuged at 100,000ꢁg for 20 min. The pellet
The microsomal fraction obtained from 30 g of the
fresh cells was incubated with 750 nmol 8-DMAN and
750 nmol NADPH in 100 mM Tris–HCl buffer (pH 8.5,
was washed twice with 100 mM Tris–HCl buffer (pH
8.5) containing 10 mM DTT and then resuspended in 5
ml of the same buffer.
ꢂ
total vol. 7.5 ml) at 30 C for 2 h. The reaction was termi-
nated by the addition of 1.5 ml of 6 M HCl, and the pro-
ducts were extracted with EtOAc (3ꢁ7.5 ml). The organic
layers were combined and concd in vacuo. The residue
was dissolved in MeOH and purified by HPLC using the
following conditions: column, Hikarisil C18 (4.6ꢁ250
mm, Asahi Chemical Ind. Co., Ltd., Japan); solvent,
CH CN/H O linear gradient solvent system, from 20%
3
.4. Protein content
Protein content was determined according to the
method of Bradford (1976).
3
.5. Enzyme reaction
3
2
CH CN to 50% CH CN in 60 min; flow rate, 1 ml/min;
3
3
ꢂ
The incubation mixture contained 150 nmol 8-dime-
oven temp., 40 C; detection, 294 nm. The fraction around
Rt 50 min was collected and evaporated in vacuo.
thylallylnaringenin dissolved in 15 ml EtOH, 150 nmol
NADPH and 60 ml of the microsomal fraction (ca. 0.5
mg of microsomal protein) in a total vol. of 150 ml. The
reaction was initiated by the addition of NADPH to the
+
EI–MS. m/z; 356 (M ), 338, 323, 295, 283, 270, 205,
192, 177, 165, 136.
ꢂ
mixture and, after incubation for 60 min at 30 C, was
terminated by the addition of 30 ml of 6 M HCl. The
reaction mixture was extracted with 150 ml of EtOAc
containing 50 nmol 1-naphthaleneacetic acid as an
internal standard. The EtOAc extract was evaporated,
dissolved in 70 ml MeOH and analyzed by HPLC.
For the investigation of the effects of different gaseous
environments, test tubes (10 ml) containing all the reac-
tion components except NADPH were sealed with septum
caps in ice-water bath, and 200 ml of N , CO/O (9:1)
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