Cytochrome P-450 dependent (+)-limonene-6-hydroxylation in fruits of caraway (Carum carvi)

Article abstract of DOI:10.1016/S0031-9422(98)00516-0

Microsomal preparations from fruits of annual and biennial forms of caraway (Carum carvi L.) catalyze the C-6 hydroxylation of (+)-limonene to (+)-trans-carveol, the key intermediate in the biosynthesis of carvone. The enzyme activities from both caraway forms had similar properties (pH optimum, K(m), cofactor requirements, etc). Both activities were dependent on NADPH and O₂, and were inhibited by N-substituted imidazoles, metyrapone, cytochrome c and CO, and thus meet many of the established criteria for cytochrome P-450-dependent mixed function oxygenases.

Full text of DOI:10.1016/S0031-9422(98)00516-0

PERGAMON
Phytochemistry 50 (1999) 243±248
Cytochrome P-450 dependent (+)-limonene-6-hydroxylation in
fruits of caraway (Carum carvi)
�
a,
Harro J. Bouwmeester *, Maurice C.J.M. Konings , Jonathan Gershenzon
a
b, 1
,
b
Frank Karp , Rodney Croteau
b
a
Research Institute for Agrobiology and Soil Fertility (AB-DLO), P.O. Box 14, 6700 AA Wageningen, The Netherlands
b
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA
Received 6 May 1998
Abstract
Microsomal preparations from fruits of annual and biennial forms of caraway (Carum carvi L.) catalyze the C-6
hydroxylation of (+)-limonene to (+)-trans-carveol, the key intermediate in the biosynthesis of carvone. The enzyme activities
from both caraway forms had similar properties (pH optimum, K , cofactor requirements, etc). Both activities were dependent
m
2
on NADPH and O , and were inhibited by N-substituted imidazoles, metyrapone, cytochrome c and CO, and thus meet many
of the established criteria for cytochrome P-450-dependent mixed function oxygenases. # 1998 Elsevier Science Ltd. All rights
reserved.
Keywords: Carum carvi; Apiaceae; Caraway; Biosynthesis; Terpenoids; ( +)-Limonene-6-hydroxylase; Carvone
1
. Introduction
example, the essential oil of caraway fruits (Carum
carvi L.) contains the monoterpenes (+)-carvone and
(+)-limonene. Because of the culinary importance of
caraway and the recent introduction of (+)-carvone
extracted from caraway fruits as an e€ective sprouting
inhibitor of potatoes (Oosterhaven, Poolman, & Smid,
1995), we have begun a detailed study of the biosyn-
thesis of (+)-carvone in caraway. In previous work
Introduction of oxygen, via hydroxylation, into the
ole®nic terpene skeletons derived from the ubiquitous
precursors geranyl diphosphate (GPP, C ), farnesyl
diphosphate (FPP, C ) and geranylgeranyl dipho-
1
0
15
sphate (GGPP, C ) is an important reaction in the
2
0
formation of the wide variety of di€erent terpenoid de-
rivatives found in the plant kingdom. Most of the en-
zymatic hydroxylations of terpene substrates described
so far involve cytochrome P-450 dependent monooxy-
genases, regardless of the size of the substrate, includ-
ing hydroxylation of the monoterpene sabinene (Karp,
Harris, & Croteau, 1987), hydroxylation of the sesqui-
terpene 5-epi-aristolochene (Hoshino et al., 1995), and
hydroxylation of the diterpene taxa-4(5),11(12)-diene
(Bouwmeester, Gershenzon, Konings,
& Croteau,
1998), we have shown that carvone formation in car-
away proceeds in an analogous fashion to the biosyn-
thesis of (�)-carvone in Mentha spicata (Gershenzon,
Ma€ei, & Croteau, 1989) (Fig. 1). In this process, a
monoterpene cyclase, (+)-limonene synthase, cyclizes
GPP to (+)-limonene. Then, the (+)-limonene formed
is hydroxylated to (+)-trans-carveol, which is sub-
+
(
Hefner et al., 1996).
Oxygenated terpenes are major components of the
essential oil of many important aromatic plants. For
sequently oxidized by an NAD utilizing terpenol de-
hydrogenase to (+)-carvone (Bouwmeester et al.,
1998). Studies of the developmental regulation of the
activities of these three enzymes suggested that (+)-
limonene-6-hydroxylation is the rate-limiting step in
the formation of (+)-carvone, based on its kinetics
and the time course of its appearance in development
�
Part 2 in the series `Biosynthesis of limonene and carvone in
fruits of caraway (Carum carvi L.)' (Bouwmeester, Gershenzon,
(
Bouwmeester et al., 1998). For this reason, and
Konings, & Croteau, in press).
*
because carvone is the commercially most important
component in caraway seed essential oil, we investi-
Corresponding author.
Present address: Max Plank Institute of Chemical Ecology, Jena,
1
Germany.
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00516-0

2
44
H.J. Bouwmeester et al. / Phytochemistry 50 (1999) 243±248
Fig. 1. Biosynthetic route to (+)-carvone.
gated the properties of the (+)-limonene-6-hydroxylase
in annual and biennial forms of caraway.
tivity remained in the supernatant upon centrifugation
at 20,000g but sedimented with the light membrane
fraction (the microsomal fraction) upon subsequent
centrifugation at 150,000g. Although some experiments
were carried out with freshly prepared microsomes,
2. Results and discussion
freezing the microsomal pellets in liquid N , and sto-
2
rage at �808C under argon seemed to enhance activity,
and, therefore, in most experiments frozen pellets were
used. These frozen microsomal pellets could be stored
for over a year at �808C without any loss of activity.
2
.1. Enzyme product, extraction conditions and stability
Caraway (+)-limonene-6-hydroxylase exhibits strict
regio- and enantiospeci®city. When the 150,000g pel-
lets of caraway fruit extracts were assayed with (+)-
limonene, the product consisted of over 95% (+)-
trans-carveol. This substance was identi®ed by GC-MS
with a chiral column using standards of (+)- and (�)-
trans-carveol obtained as described by Bouwmeester et
al. (1998).
Optimization of the extraction procedure was car-
ried out with annual caraway. Substantial (+)-limo-
nene-6-hydroxylase activity was obtained when frozen
fruits were ground on ice in the presence of a cold
Hepes bu€er, pH 7.5, containing glycerol, polyvinylpo-
lypyrrolidone (PVPP), reducing agents, protease inhibi-
tors and other protectants. When seeds were ground in
2.2. Characterization of limonene-6-hydroxylase activity
The limonene-6-hydroxylase activity of annual car-
away was ca. 3-fold higher than that of the biennial
form (Table 1). This di€erence in activity is due to a
di€erence in the age of the fruits used for extraction
rather than a fundamental di€erence between the two
forms (Bouwmeester et al., 1998). The hydroxylases
from both forms required NADPH and O for cataly-
2
sis, and NADH did not support activity (Table 1). The
presence of an NADPH-regenerating system slightly
stimulated hydroxylation activity, but incubation with-
out O , under an Ar atmosphere or with an O -scaven-
liquid N (without bu€er), there was a complete loss
2
2
2
of enzyme activity. Polyvinylpyrrolidone was instru-
mental in preserving enzyme activity with the insoluble
form (PVPP) at equal tissue weight being more e€ec-
tive than the soluble form (PVP-40) at 3±4% (w/v).
ging system decreased activity by 85±96% (Table 1).
Microsomal NADPH- and O -requiring hydroxylation
2
reactions also depend on the activity of an NADPH-
cytochrome P-450 reductase for the electron transfer
from NADPH to the cytochrome P-450 oxygenase.
The ¯avin moiety of this reductase is often labile and
inclusion of FAD and FMN during preparation of
microsomal pellets or in the assay generally enhances
hydroxylase activity (Mihaliak, Karp, & Croteau,
1993). However, in this study the omission of FAD
and FMN from the extraction bu€er did not have a
negative e€ect on (+)-limonene-6-hydroxylase activity
as long as ¯avins were present during the assay. When
FAD and FMN were omitted from the assay, (+)-
limonene-6-hydroxylase activity decreased by over
50% (Table 1).
A number of compounds known to inhibit cyto-
chrome P-450 catalyzed oxygenation reactions were
tested to examine the possible involvement of cyto-
chrome P-450 in the hydroxylation of (+)-limonene in
caraway. Clotrimazole and miconazole have been pre-
viously shown to be e€ective inhibitors of cytochrome
Among reducing agents, the use of NaHSO was more
3
e€ective in preserving enzyme activity than sodium
diethyldithiocarbamate. Omitting leupeptin, EGTA,
catalase and glutathione reduced the activity by almost
50% although the e€ect of the individual compounds
of this mixture was not assessed. Following grinding,
the extract was treated with polystyrene resin (XAD-4)
to adsorb phenolics and terpenes. The amount of
XAD-4 used strongly a€ected the recovery of hy-
droxylase activity, with maximal activity obtained with
0.5 g XAD-4 per g of tissue. When no XAD-4 was
employed, activity was 75% of maximum, while levels
�
1
of 1 g g
resulted in only 50% of maximal activity.
After XAD-4 treatment, sonication for several minutes
on ice improved hydroxylase activity by ca. 60%.
Di€erential centrifugation at 20,000 and 150,000g
was routinely used for the preparation of microsomal
pellets since almost 90% of the total hydroxylase ac-

H.J. Bouwmeester et al. / Phytochemistry 50 (1999) 243±248
245
Table 1
Requirements for caraway (+)-limonene-6-hydroxylase activity. All values are the mean of at least two independent experiments
a
b
Assay conditions
Annual caraway
Biennial caraway
�
1
pkat g fruit (dry wt))
� 1
(
(%)
(pkat g fruit (dry wt))
(%)
c
+RS (control)
NADPH+FAD, FMN+O
Control-RS
2
218
206
100
94
0
75.2
63.3
0.71
5.67
7.69
30.5
100
84
1
Control-NADPH� RS +NADH
0.0
31.9
9.78
89.7
Control-O
Control-O
2
(Ar atmosphere)
(O scavenging system )
15
4
8
d
2
2
10
45
Control-FAD, FMN
41
a
Extracted in bu€er without FAD, FMN.
b
Extracted in bu€er with FAD, FMN.
RS =NADPH regenerating system (5 mM glucose-6-phosphate, 1 i.u. glucose-6-phosphate dehydrogenase).
c
d
2
O scavenging system (10 i.u. of glucose oxidase, 6 mmol b-D-glucose and 1300 i.u. catalase).
P-450 enzymes (Karp et al., 1987; Karp, Mihaliak,
Harris, & Croteau, 1990; Hallahan, Dawson, West, &
Wallsgrove, 1992). In caraway, clotrimazole was ca.
1995). Cytochrome c inhibits cytochrome P-450 cata-
lyzed reactions by acting as an alternative electron
acceptor for reducing equivalents from cytochrome P-
450 reductase (Mihaliak et al., 1993). Cytochrome c
strongly inhibited caraway (+)-limonene-6-hydroxyl-
ase activity (I50 < 10 mM) (Table 2) suggesting involve-
ment of the P-450 system.
200±300-fold more e€ective in inhibiting limonene-6-
hydroxylation than was miconazole (Table 2). Sheet,
Mason, Wise, & Estabrook (1986) and Karp et al.
(
1990) showed that these N-substituted imidazoles may
be useful for distinguishing among di€erent forms of
cytochrome P-450. For example, enzymes that catalyze
the oxygenation of a single substrate at di€erent pos-
itions may be di€erentially a€ected by the various in-
hibitors (Karp et al., 1990). The present work shows
that these inhibitors can also distinguish among
enzymes whose chiral speci®city di€ers. Whereas (�)-
limonene-6-hydroxylase from M. spicata was inhibited
equally by miconazole and clotrimazole (Karp et al.,
The inhibition of enzyme activity by CO and its
reversal by blue light are another indication of the
involvement of cytochrome P-450. CO inhibited the
limonene-6-hydroxylase activity of biennial caraway in
a concentration dependent manner (Table 3), while, in
annual caraway, a 100% CO atmosphere completely
inhibited limonene-6-hydroxylation (data not shown).
However, blue light reversal was not clearly demon-
strated because blue light strongly decreased enzyme
activity in control assays (Table 3), which can prob-
ably be attributed to ¯avin or chlorophyll-dependent
oxygen radical generation (Karp et al., 1987).
1990), (+)-limonene-6-hydroxylase from caraway was
inhibited 200±300-fold more by clotrimazole than
miconazole (Table 2). Another inhibitor often used to
demonstrate the involvement of cytochrome P-450 is
metyrapone. At low concentrations, metyrapone
slightly stimulated limonene-6-hydroxylase activity and
high concentrations were required for inhibition
Nevertheless, at a high CO:O ratio, blue light reduced
2
the enzyme activity less than expected on the basis of
photoinhibition alone.
The pH optima for the two hydroxylases in Tris
bu€er were similar, 7.4 for annual and 7.2 for biennial
caraway, with estimated half-maximal activities at a
pH of ca. 6.3 and 8.4 for both enzymes. Enzyme ac-
tivities in Mes and Bis±Tris bu€ers were similar, but
(
Table 2). This is in agreement with the sensitivity to
metyrapone of the (�)-limonene hydroxylases (Karp et
al., 1990), geraniol-10-hydroxylase (Hallahan et al.,
0
992) and naringenin 3 -hydroxylase (Doostdar et al.,
1
Table 2
Inhibition of caraway (+)-limonene-6-hydroxylases
Table 3
Carbon monoxide inhibition and blue light reversal of (+)-limonene-
6
-hydroxylase activity from biennial caraway
a
Inhibitor
I
50 (mM)
�
1
Enzyme activity (pkat g fruits (dry wt))
CO:O
2
annual caraway
biennial caraway
dark
blue light
9.13
Clotrimazole
Miconazole
Metyrapone
Cytochrome c
< 1
47.7
0.34
103
air
53.7
56.6
37.4
22.3
6.62
1516
<10
1459
2.87
1
4
6
8
:9
:6
:4
:2
7.96
a
Inhibitor concentration for half-maximal activity determined from
measurements of at least four separate concentrations.

2
46
H.J. Bouwmeester et al. / Phytochemistry 50 (1999) 243±248
much lower than in Tris bu€er. The shape of the pH
curve is typical for cytochrome P-450 enzymes
solubilization and puri®cation of the cytochrome P-
450 dependent enzymes, it is still usually dicult to
purify these proteins and isolate the corresponding
gene using amino acid sequence information for the
puri®ed protein. Recent successes in the cloning of P-
450 genes have resulted from a PCR strategy using pri-
mers based on the conserved regions in their sequences
(Meijer et al., 1993; Schuler, 1996). As this approach
often yields a multitude of P-450 clones, a biochemical
characterization of the enzymes using the information
presented in this communication will be necessary to
identify the gene of interest.
(
1
Mihaliak et al., 1993; Madyastha, Meehan, & Coscia,
976) and the pH optima found are similar to pH
optima reported for other cytochrome P-450 hydroxy-
lases such as 7.4 for sabinene hydroxylase (Karp et al.,
1
987) and (�)-limonene C-3 and C-6 hydroxylase
(
(
Karp et al., 1990), 7.0 for camphor hydroxylase
0
Funk & Croteau, 1993) and 7.4±7.6 for naringenin 3 -
hydroxylase (Doostdar et al., 1995).
Additional evidence for the involvement of cyto-
chrome P-450 can be obtained from spectral data, i.e.
by carbon monoxide di€erence spectroscopy (Omura
&
Sato, 1964). However, CO di€erence spectra could
not be obtained with microsomal preparations from
caraway fruits probably because of interfering pig-
ments. Researchers commonly encounter diculties
doing spectral studies on membrane suspensions from
green plant tissues due to the presence of green pig-
ments (Hallahan et al., 1992).
4. Experimental
4.1. Plant materials
Annual caraway (Carum carvi L.) var. `Karzo' was
grown in the ®eld, essentially as described
(
Bouwmeester & Smid, 1995). Biennial caraway var.
`Bleija' was grown in a greenhouse as described
Bouwmeester et al., 1998). Batches of unripe fruits of
2.3. Kinetics
(
Plots of limonene-6-hydroxylase activity versus limo-
both caraway forms were harvested, frozen in liquid
nene concentration gave rise to typical hyperbolic sat-
uration curves (Fig. 2). Using non-linear regression for
kinetic analysis, yielded apparent Km values of 11.4
mM for annual and 14.9 mM for biennial caraway,
similar to values reported for other terpenoid hydroxy-
lases such as geraniol-10-hydroxylase (15 mM
N and stored at �808C until further use. Fruit dry
2
matter percentage was determined by weighing a sub
sample of ca. 0.5±1.0 g (fr. wt) of fruits before and
after drying overnight at 1058C.
4.2. Preparation of microsomes
(
Hallahan et al., 1992)) and (�)-limonene C-3, C-6 and
C-7 hydroxylases (18, 20 and 21 mM) (Karp et al.,
During enzyme isolation and preparation of the
assays, all operations were carried out on ice or at
1990).
48C. Samples of 2 g of the frozen fruits were ground
in a pre-chilled mortar and pestle in 10 ml of a pre-
3. Conclusion
chilled bu€er containing 50 mM Hepes (pH 7.5), 20%
(
v/v) glycerol, 50 mM sodium ascorbate, 50 mM
NaHSO , 5 mM MgCl , 2.5 mM EDTA, 2.5 mM
EGTA, 5 mM DTT, 5 mM FAD, 5 mM FMN, 0.5
The present results con®rm that (+)-limonene in
3
2
caraway is hydroxylated at the 6-position to (+)-trans-
carveol by a cytochrome P-450 dependent monooxy-
genase in analogous fashion to the 6-hydroxylation of
�
1
� 1
mM glutathione, 2 mg ml BSA, 5 mg ml leupeptin
�
1
and 25 i.u. ml
catalase slurried with 1 g PVPP and
(
�)-limonene to (�)-trans-carveol in M. spicata
some puri®ed sea sand. During grinding, additional
aliquots of bu€er (without PVPP and sea sand) were
added to a total volume of ca. 60 ml. The homogenate
was transferred to a small beaker containing 1 g of
polystyrene resin (XAD-4), sonicated for 4 min in 10 s
pulses (on ice), stirred carefully for 12 min and then
®ltered through cheesecloth. The ®ltrate was centri-
fuged at 20,000g for 20 min (pellet discarded) and then
at 150,000g for 90 min. Microsomal pellets were either
(
Gershenzon et al., 1989; Karp et al., 1990).
Cytochrome P-450 dependent hydroxylases have also
been implicated in the hydroxylation of other monoter-
penoids such as sabinene (Karp et al., 1987), geraniol
(
Hallahan et al., 1992; Meijer, Souer, Verpoorte, &
Hoge, 1993) and camphor (Funk & Croteau, 1993).
The enzymes from the biennial and annual forms of
caraway are very similar in properties, including Km,
pH optimum, cofactor requirements and the degree of
inhibition by cytochrome P-450 inhibitors.
As (+)-limonene-6-hydroxylation was shown to
limit carvone formation in caraway (Bouwmeester et
al., 1998), gene isolation and over expression may
enable an increase in the carvone content of this im-
portant spice plant. However, despite progress in the
used directly for assays or frozen in liquid N
stored under argon at �808C.
and
2
4.3. Enzyme assays
Microsomal pellets were resuspended in a small
volume of assay bu€er containing 50 mM Tris (pH 7.2

H.J. Bouwmeester et al. / Phytochemistry 50 (1999) 243±248
247
Fig. 2. (+)-Limonene hydroxylase activity as function of substrate concentration and double reciprocal plots of the data (insert) for (A) the
2
annual and (B) biennial form of caraway. (A) y= 233.26x/(11.35+ x), R =0.86 (B) y=78.91x/(14.87+ x), R = 0.93.
2
for biennial, pH 7.4 for annual), 20% (v/v) glycerol, 1
�
enzyme activities of both caraway forms were linear
for over 60 min. Control assays that had been boiled
for 5 min showed no enzymatic activity. After incu-
1
mM EDTA, 2 mM DTT, 1 mg ml
leupeptin, 5 mM
FAD and 5 mM FMN using a glass rod and te¯on
potter and then diluted to the desired concentration
using the same bu€er. To determine pH optima, buf-
fers of 50 mM Mes and 50 mM Bis±Tris were also
used. One ml of this microsomal suspension (in stan-
dard assays: 6.9±8.3 mg protein) was incubated in a
bation for 1 h at 308C, 1 ml of Et O was added and
2
the tube vigorously mixed and then stored at �208C
until further analysis. To assess the levels of limonene
and carveols initially present, control assays were run
in which the reaction was stopped immediately after
9
ml te¯on-lined screw cap vial and the reaction
substrate addition by adding 1 ml Et O and vigorous
2
started by the addition of 1 mM NADPH, an
NADPH-regenerating system (5 mM glucose-6-phos-
phate, 1 i.u. glucose-6-phosphate dehydrogenase) and
mixing. For analysis, 25 nmol camphor was added to
the reaction mixture as an internal standard. The reac-
tion mixtures were thawed, vigorously mixed and
200 nmol (+)-limonene (5 ml of a hexane stock). The
brie¯y centrifuged to separate phases. The Et O phase
2
assays were performed in duplicate. In routine assays,
was then transferred to another vial, and the water

2
48
H.J. Bouwmeester et al. / Phytochemistry 50 (1999) 243±248
�
1
layer re-extracted with another 1 ml portion of Et O.
2
9 mmol g
seed (dwt) for annual caraway). These
The combined Et O extracts were decolorized with
2
could only partially be removed by increasing the
amount of XAD-4 used during extraction, which was
however undesirable as higher amounts of XAD-4
decreased the yield of limonene-6-hydroxylase activity.
Hence, kinetic analyses were carried out by ®rst dilut-
ing the enzyme preparation, and then adding ad-
ditional limonene to obtain a range of substrate
concentrations.
activated charcoal, washed with 1 ml of water and,
after centrifugation, passed over a short column of
silica gel overlaid with anhydrous MgSO . The water
4
phase was re-extracted with another 1 ml portion of
Et O which was also passed over the MgSO /silica gel
2
4
column. After 500 ml of hexane were added, the
extracts were concentrated to ca. 500 ml using a cen-
trifugal evaporator and, subsequently, a stream of N2.
Samples were analyzed for limonene, camphor, cis-
and trans-carveol and carvone using GC-MS in the
selected ion monitoring mode: for limonene m/z 68,
4.4. Other analytical procedures
Attempts to obtain CO-di€erence spectra were car-
ried out according to (Omura and Sato, (1964) with
sodium dithionite as reductant. Because BSA was
included in the extraction bu€er, microsomal protein
levels were determined using the micro-assay protocol
of the Coomassie Plus Protein Assay (Pierce) and BSA
as protein standard after resuspension of the pellet in
a bu€er without BSA, an additional 150,000  g cen-
trifugation step and resuspension of the resulting pellet
in water.
93, 136; for camphor: m/z 81, 95, 152; for trans-carveol
m/z 84, 109, 152; for cis-carveol m/z 84, 109, 134; for
carvone m/z 82, 108, 150 on a HP 5890 series II GC
and HP 5972A Mass Selective Detector. The GC was
equipped with a HP-5MS column (30 m  0.25 mm
id  0.25 mm df). The injection port (splitless mode),
interface and MS source temp. were 175, 290 and
1808C, respectively. The He inlet pressure was con-
trolled by Electronic Pressure Control to achieve a
�
1
constant column ¯ow of 1.0 ml min
during the fol-
lowing oven program: initial temp. 458C for 1 min,
�
1
ramp of 108C min
to 2208C and a ®nal time of 5
min. Ionization potential was set at 70 eV. The com-
pounds were quanti®ed using an external standard
mixture with known concentrations of authentic refer-
ence compounds that was measured under the same
conditions.
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&
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2
vials were ¯ushed with argon immediately after ad-
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2
essentially as described (Karp et al., 1987). Assays
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reaction mixture (containing substrate and cofactor)
was slowly bubbled before incubation with 45 ml of
the gas mixture through a needle inserted through the
(vented) septum. Blue light was obtained by passing
the beam of a tungsten lamp (250 W) through 10%
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4
2
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approximately 3 mmol g seed (dwt) for biennial and