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Plant Physiol. (1998) 117: 1381-1392
(+)-Germacrene A Biosynthesis
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The leaves and especially the roots
of chicory (Cichorium intybus L.) contain high
concentrations of bitter sesquiterpene lactones such as the guianolides
lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and
germacranolides are present in smaller amounts. Their postulated
biosynthesis through the mevalonate-farnesyl diphosphate-germacradiene
pathway has now been confirmed by the isolation of a (+)-germacrene A
synthase from chicory roots. This sesquiterpene cyclase was purified
200-fold using a combination of anion-exchange and dye-ligand
chromatography. It has a Km value of 6.6 µM, an estimated molecular mass of 54 kD, and
a (broad) pH optimum around 6.7. Germacrene A, the enzymatic product,
proved to be much more stable than reported in literature. Its
heat-induced Cope rearrangement into (
)-
-elemene was utilized to
determine its absolute configuration on an enantioselective gas
chromatography column. To our knowledge, until now in sesquiterpene
biosynthesis, germacrene A has only been reported as an (postulated)
enzyme-bound intermediate, which, instead of being released, is
subjected to additional cyclization(s) by the same enzyme that
generated it from farnesyl diphosphate. However, in chicory germacrene
A is released from the sesquiterpene cyclase. Apparently, subsequent oxidations and/or glucosylation of the germacrane skeleton, together with a germacrene cyclase, determine whether guaiane- or eudesmane-type sesquiterpene lactones are produced.
The sprouts of chicory (Cichorium intybus L.), a
vegetable (French endive) grown in the dark, are known for their
slightly bitter taste originating from sesquiterpene lactones. The tap roots of chicory in particular are extremely bitter due to these components with antifeedant properties (Rodriguez et al., 1976 The average sesquiterpene lactone content is measured at 0.42% dry
weight in the roots and 0.26% in the leaves (Rees and Harborne, 1985
It is assumed that both the guaiane- and eudesmane-type lactones
originate from a common germacrane precursor that is formed via the
acetate-mevalonate-FPP pathway by a germacrene synthase, an enzyme
belonging to the group of sesquiterpene cyclases (Herz, 1977
Biosynthetic studies with a hairy-root culture of blue-flowered lettuce
supplied with 13C-labeled precursors of secondary
plant metabolism (acetate and mevalolactone) seem to confirm this
acetate-mevalonate-FPP-germacradiene pathway. From the patterns of
13C-enrichment in the produced guaianolides, Song
et al. (1995) Sesquiterpene cyclases catalyze the conversion of FPP to over 200 different cyclic skeletons, and a growing number of these enzymes have
been isolated and characterized in recent years. cDNA sequences (Cane,
1990 A problem in studying germacrene synthases may be the reported
instability of all four known germacrenes. Germacrene A (Fig. 3, 7) is reported to be
particularly susceptible to proton-induced cyclizations toward
The aim of our research was to identify the germacrane intermediate
involved in the sesquiterpene lactone biosynthesis of chicory and to
isolate and characterize the sesquiterpene cyclase responsible for
its formation.
Fresh roots of cultivated chicory (Cichorium intybus L. cv Focus) were harvested during late summer and obtained from a
grower in Veenendaal, The Netherlands. Roots of wild chicory were
collected in October in the forelands of the Rhine near Wageningen. The chicory roots were cut into small pieces, frozen in liquid nitrogen, and stored at Enzyme Isolation and Assay I
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Rees
and Harborne, 1985; van Beek et al., 1990). In the past, these roots
were roasted and used as a coffee substitute; now they are regarded as
a waste product of chicory cultivation. About 100,000 tons of chicory
roots are produced annually in The Netherlands, but because of their
bitter taste, it is not possible to use them as cattle feed. In
searching for a way to enhance the value for this waste product, we
have been investigating the bitter sesquiterpene lactones (van Beek et
al., 1990; Leclercq, 1992), and we are interested in the biosynthesis
of these compounds because it involves stereoselective oxidizing
enzymes that might be useful as catalysts in organic syntheses.
).
The three major sesquiterpene lactones in chicory are the guaianolides
lactucin (see Fig. 1, 1),
8-deoxylactucin (Fig. 1, 2), and lactupicrin (Fig. 1,
3), which are present in both leaves and roots of chicory
(Pyrek, 1985; van Beek et al., 1990). The two eudesmanolides sonchuside
C (Fig. 1, 4) and cichoriolide A (Fig. 1, 5) are
also present, together with the germacranolides sonchuside A and
cichorioside C (Seto et al., 1988).
View larger version (15K):
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Figure 1.
The three major guaianolides of chicory, lactucin
(1), 8-deoxylactucin (2), and lactupicrin
(3), and its two eudesmanolides, sonchuside C (4)
and cichoriolide A (5).
; Bohlman
and Zdero, 1978; Fischer, 1990; Song et al., 1995). Whether this common
germacrane precursor is transformed into a guaiane skeleton or a
eudesmane skeleton would depend on the position of enzyme-mediated
epoxidations. A germacrene
C4-C5-epoxide would lead to
a guaiane, whereas a germacrene
C1-C10-epoxide would lead to a eudesmane (Brown et al., 1975; Teisseire, 1994; Piet et al., 1995b). For this reason we postulated that, apart from the oxidizing enzymes, two different cyclizing enzymes are involved in the
biosynthesis of the guaianolides and eudesmanolides: an enzyme that
cyclizes FPP to a germacrane skeleton, and a separate enzyme that
cyclizes the germacrane skeleton to a guaiane or eudesmane skeleton
(Fig. 2) (Piet et al., 1995b, 1996).
View larger version (14K):
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Figure 2.
A simplified scheme (without oxidative steps) for
the formation of eudesmane- and guaiane-type compounds involves two
cyclizing enzymes, a germacrene synthase and a germacrane cyclase.
Literature suggests that either germacrene A or germacrene B is the
germacrane intermediate.
deduced that the C12 and the
C13 atoms of the germacrane intermediate are
chemically not identical; this indicates the formation of either
germacrene A (7) or germacrene B. Formation of germacrene B
would be supported by the existence of
C8-oxygenated sesquiterpene lactones such as
lactucin and lactupicrin, because in germacrene B the
C8 position is activated for allylic oxidations
(Fischer, 1990).
; McCaskill, 1997) are available, and protein crystal structures
have been published recently (Lesburg et al., 1997; Starks et al.,
1997). Although germacrenes, especially germacrene D, are important
constituents of many essential oils, to our knowledge, no germacrene
synthase has so far been described. The biosynthesis of germacrene C by
a homogenate of immature seeds of Kadsura japonica has been
reported (Morikawa et al., 1971), as well as the partial
purification of a synthase for -selinene (Belingheri et al., 1992),
a germacrene A-related compound, from the outer peels of
Citrofortunella mitis.
-selinene (Fig. 3, 8) and -selinene (Fig. 3,
9) on silica gel, or to Cope rearrangement toward -elemene (Fig. 3, 10), even during freezer storage
(Weinheimer, 1970; Bowers et al., 1977; Teisseire, 1994). Germacrene A
itself has often been postulated as an intermediate (bound to the
sesquiterpene cyclase) in the biosynthesis of patchoulol and
phytoalexins, such as aristolochene, 5-epi-aristolochene, capsidiol,
debneyol, and vetispiradiene (Threlfall et al., 1988; Hohn et al.,
1989; Whitehead et al., 1989; Beale, 1990; Cane, 1990; Munck and
Croteau, 1990; Cane et al., 1993; Back and Chappell, 1995).
View larger version (10K):
[in a new window]
Figure 3.
The reported high sensitivity of germacrene A
(7) to heat and slightly acidic conditions gives
rearrangement toward -elemene (10), respectively,
cyclization toward -selinene (8) and -selinene
(9). Selina-4,11-diene (11) would be another
acid-induced cyclization product.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
80°C. Unlabeled FPP was obtained from Sigma in a
solution of 70% (v/v) methanol in 10 mM aqueous ammonium
hydroxide. The solvent was evaporated in vacuo using a Gyrovap GT
(Howe, Oxon, UK), and a 10 mM FPP stock solution was
prepared with 50% (v/v) ethanol in 200 mM aqueous ammonium
bicarbonate. [1-3H]FPP dissolved in a solution of 50%
(v/v) ethanol with 100 mM aqueous ammonium bicarbonate
(16.0 Ci mmol
1, 200 µCi
mL1) was purchased from Amersham.
1), using an overlay of 1 mL of pentane
(assay I). As a control, both types of incubations were also
performed with supernatant that had been boiled for 5 min. Incubations
were stopped by vigorous shaking. For analysis, the pentane
phase was filtered through a dimethyl chlorosilane-treated glass wool
(Chrompack, Bergen op Zoom, The Netherlands) plugged Pasteur pipette
that contained 0.90 g of aluminum oxide (grade III) and a little
anhydrous magnesium sulfate. The extraction was repeated with 1 mL of
20% (v/v) ether in pentane, and the aluminum oxide column was washed
with this extract and an additional amount of 1.5 mL of 20% (v/v)
ether in pentane. The combined pentane eluate contained sesquiterpenoid hydrocarbons that do not bind to aluminum oxide under these conditions. The same assay was then reextracted with an equal portion of ether, and
the ether extracts were passed through the same column. At the end of
this extraction-filtration procedure, the column was rinsed with 1.5 mL
of ether to ensure a complete elution of (oxygenated) products. The
separately collected pentane/ether and ether phases were carefully
concentrated to approximately 50 µL under a stream of nitrogen. Both
the pentane and ether (diethyl ether) were redistilled before use in
the filtration-extraction procedure described above.
Identification of Sesquiterpenoid Products
Before the concentrated extracts of the [3H]FPP incubated assays were analyzed by radio-GC, 1 µL of a sesquiterpene standard was added containing (each 1 mg mL1 in pentane) germacrene B
(prepared according to the method of Piet et al., 1995b), 
-elemene
(prepared from germacrene B by heating at 160°C under argon for
16 h), nerolidol, and farnesol. In later experiments this
sesquiterpene standard was replaced by either 5 µL of an alkane set
(n = 7-22, 1 mg mL1 each in
pentane) to calculate Kovats' indices, or 5 µL of a liverwort (Frullania tamarisci) extract containing germacrene A as one
of its major constituents (Hardt et al., 1995; W.A. König,
unpublished results). Radio-GC analysis was performed on a Carlo-Erba
4160 (Milano, Italy) series gas chromatograph coupled to a RAGA
93 radioactivity detector (5-mL counting tube, Raytest, Straubenhart, Germany). The GC was equipped with a CP-WAX 52 column (25 m × 0.32 mm, film thickness of 0.25 µm) and programmed at 5°C
min1 from 70°C to 210°C using a helium flow
rate of 2.7 mL min1. Samples of 1 µL were
injected in the cold on-column mode. The compounds eluting from the
column were split in a ratio of 3:1 between the radioactivity detector
and a flame-ionization detector at 210°C. Before entering the
radioactivity detector, eluted compounds were quantitatively reduced by
addition of hydrogen at 3 mL min1 and passage
through a conversion reactor filled with platinum chips at 800°C.
After reduction, methane was added as a quenching gas to give a total
flow of 36 mL min1 through the counting tube of
the radioactivity detector. The incubations with unlabeled FPP were
analyzed by GC-MS using a 5890 series II gas chromatograph
(Hewlett-Packard) equipped with a mass selective detector (model 5927A,
Hewlett-Packard) and a capillary HP-5MS column (30 m × 0.25 mm,
film thickness of 0.25 µm) at a helium flow rate of 0.969 mL
min1. The splitless injection of the 1-µL
sample was initially done at an injection port temperature of 210°C,
and at 150°C in later experiments because of the sensitivity of
germacrenes to high temperatures. After an initial temperature of
55°C for 4 min, the column was programmed at 5°C
min1 to 210°C. The mass spectra were recorded
at 70 eV scanning from 30 to 250 atomic mass units. MS data were
compared with those recorded from compounds present in the natural oil
of Mentha mirennea (-elemene [Fig. 3,
10]; Maat et al., 1992) and the extract of F. tamarisci (germacrene A [Fig. 3, 7], -selinene
[Fig. 3, 8], -selinene [Fig. 3, 9], and
selina-4,11-diene [Fig. 3, 11]; W.A. König,
unpublished results).
Determination of the Absolute Configuration of Germacrene A
The absolute configuration of germacrene A (Fig. 3, 7) was determined by means of its Cope rearrangement to-elemene (Fig.
3, 10), a reaction that occurs with retention of stereochemical configuration at C7 (Weinheimer et al.,
1970; Takeda, 1974; March, 1992). The GC-MS was essentially used as
described above, but with an injection port temperature of 250°C to
induce the rearrangement of enzymatically produced germacrene A. The oven temperature was programmed to 45°C for 4 min followed by a ramp
of 2°C min1 to 170°C, and spectra were
recorded in the selected ion-monitoring mode (m/z 121, 147, and 189). The apparatus itself was equipped with a 25-m (0.25-mm i.d.)
heptakis(6-O-TBDMS-2,3-di-O-methyl)--cyclodextrin (50% in OV17) column that is able to separate the enantiomers of
racemic -elemene (König et al., 1994). A racemic -elemene standard was isolated from a hydrodistillate of the liverwort Frullania macrocephalum (gift of Dr. L. Kraut, University of
Saarbrücken, Germany), and the elution order of its enantiomers
was determined with a ()--elemene standard (König et al.,
1994). To substantiate correct identification of the -elemene
enantiomer derived from chicory germacrene A, racemic -elemene was
co-injected with enzymatically produced germacrene A at injection port
temperatures of 150°C and 250°C.
Sesquiterpene Cyclase Assay II and Protein Determination
For routine determination of germacrene A synthase activity, 10 µL of sample was added to an Eppendorf vial with 90 µL of buffer C (0.1% [v/v] Tween 20 in buffer B) and incubated at 30°C with 20 µM [3H]FPP (50 Ci mol1). The reaction mixture was overlaid with 1 mL of hexane to trap formed, labeled olefins (assay II). After 30 min
the vial was vigorously mixed and cooled to stop the reaction, then it
was briefly centrifuged to separate phases. In the hexane phase, 750 µL was transferred to a new Eppendorf vial containing 40 mg of silica
(0.06-0.2 mm) to bind farnesol produced from FPP by phosphohydrolases. After mixing and centrifugation, 450 µL of the hexane layer was removed for scintillation counting in 4.5 mL of Ultima Gold cocktail (Packard, Meriden, CT). Protein concentrations were determined using the microassay protocol of the Coomassie Plus Protein Assay (Pierce) and BSA as a protein standard. Mono-Q fractions containing Tween 20 were desalted to 50 mM ammonium bicarbonate using
a HiTrap desalting column and assayed by the Micro BCA Protein Assay
(Pierce).
Sesquiterpene Cyclase Purification
Cellular extracts and enzyme preparations were kept on ice throughout the purification. The purification was started by making a 100,000g supernatant as described above, but with a buffer containing 25 mM Mopso (pH 7.0), 25 mM sodium meta-bisulfite, 25 mM ascorbic acid, 10 mM MgCl2, and 2 mM DTT (buffer D). A column (Ø 2.5 cm) of 25 g of DEAE (preswollen DE52, Whatman) suspended in 150 mL of buffer containing 150 mM Mopso (pH 7.0), 100 mM MgCl2, and 20 mM sodium meta-bisulfite was prepared and washed at 1.6 mL min1 with 150 mL of 2 mM
sodium meta-bisulfite in buffer B (buffer E). Seventy-five
milliliters of the 100,000g supernatant was loaded onto this
column and washed with another 100 mL of buffer E to remove unbound
proteins. A 100-mL gradient of 0 to 0.5 M KCl in buffer E
was used to elute sesquiterpene cyclase activity. Fractions containing
sesquiterpene cyclase activity were pooled and desalted to buffer B,
after which glycerol was added to a final concentration of 30% (v/v).
The enzyme preparation was frozen in liquid nitrogen and stored at
80°C in 1-mL aliquots. One aliquot was tested for the nature of its
enzymatic sesquiterpenoid product, whereas the others served as a stock
for further purification steps.
1 Reactive Green 5 that had been rinsed twice
with an equal volume of buffer B. The column was equilibrated with 15 mL of buffer B, and an aliquot of DEAE-purified cyclase (thawed and
warmed up to room temperature) was applied to this column at 0.5 mL
min1. Unbound proteins were washed off the
column with buffer B while monitoring the
A280; the sesquiterpene cyclase was eluted
using a one-step gradient of 1.5 M KCl in buffer B. Sesquiterpene cyclase activity containing fractions were combined,
desalted to buffer C with an Econo-Pac 10DG column, and applied to a
Mono-Q fast-protein liquid chromatography column (HR5/5, Pharmacia
Biotech) previously equilibrated with buffer C. The column was washed
with 4 mL of buffer C at 0.75 mL min1, after
which bound proteins were eluted with a 26-mL gradient of 0 to 0.66 M KCl in buffer C. Fractions were assayed, and those containing enzyme activity were tested for the nature of their sesquiterpenoid product(s). The fraction (0.75 mL) containing the
highest amount of germacrene A synthase activity was used to determine
the Mr and Km
(see below). After each purification step the purification was
visualized by SDS-PAGE using preprepared 10% (w/v) polyacrylamide gels
(Bio-Rad) according to the manufacturer's instructions. Gels were
stained using a silver-staining kit (Pharmacia Biotech).
Km, pH Optimum, and Mr Determination
Before determining the Km of the chicory germacrene A synthase, assay II (with buffer C) was checked for its linearity using Mono-Q-purified enzyme. When the enzyme was diluted with an equal amount of buffer C, the assay was linear during the first 40 min (r2= 0.987) at a concentration of 2 µM FPP. When undiluted Mono-Q-purified enzyme was used, enzyme activity was twice as high and linear during 30 min (r2= 0.963) (Fig. 8A). For the kinetics study, enzyme activity was determined in the range of 0.5 to 80 µM FPP (enzyme diluted twice in buffer C). The pH optimum was determined with DEAE-purified germacrene A synthase in the pH range of 4.0 to 9.0 using the protocol of assay II and 5 µL of enzyme. For pH values of 4.0 to 5.5, 5.5 to 6.5, and 7.5 to 9.0, NaAc, Mes, and Tris-HCl, respectively, were used instead of Mopso. pH experiments were carried out in duplicate and in both the presence and the absence of 0.1% Tween 20. The Mr of the germacrene A synthase from chicory was estimated by exclusion chromatography on a Superdex 75 column (HR10/30, Pharmacia Biotech) in buffer C. The column was calibrated at 0.5 mL min1 with cyt
c (12.4 kD), RNase A (13.7 kD), -chymotrypsinogen (25.0 kD), ovalbumin (45.0 kD), and BSA (67.0 kD), all purchased from Sigma.
The column was loaded with 200 µL of Mono-Q-purified germacrene A
synthase, and fractions of 0.5 mL were assayed for their sesquiterpene cyclase activity.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Detection of the (+)-Germacrene A Synthase Activity in Chicory Roots
A 100,000g supernatant was prepared from both cultivated and wild chicory and incubated with [3H]FPP. The incubations were extracted subsequently with pentane and ether; the extracts were analyzed by radio-GC after being passed over a short aluminum oxide column. The pentane extracts of both types of plant material revealed one radioactive product, which in the ether extracts was accompanied by farnesol, a result of aspecific phosphohydrolase activity. The product peak did not coincide with that of germacrene B nor that of-elemene. The unknown product and farnesol were not present when the
supernatant was boiled before incubation, and the amount of product was
raised after the DEAE purification step in which almost all
phosphohydrolase activity was discarded (Fig.
4).
|
-selinene (Fig. 3,
8), -selinene (Fig. 3, 9), and
selina-4,11-diene (Fig. 3, 11), typical acid
(silica)-induced cyclization products of germacrene A. The Kovats'
index of our enzymatic product was 1737, which matches the value
reported for germacrene A (1734; M.H. Boelens [1995] database
Essential Oil, version 4.1, The Netherlands). The final proof for the
identity of the unknown product was obtained by co-injection of an
extract from the liverwort F. tamarisci, which contains
germacrene A (Fig. 4).
-elemene (Fig. 3, 10) almost completely during the
measurement. This problem was overcome by lowering the GC injection
port temperature from 210°C to 150°C. At this lowered temperature,
almost no Cope rearrangement of germacrene A occurred, although the
germacrene A peak broadened significantly and was preceded by a
"hump" in the baseline.
Purification of the (+)-Germacrene A Synthase from Chicory Roots
Characterization of the (+)-Germacrene A Synthase
The sole enzymatically produced sesquiterpenoid product in
incubations of a 100,000g chicory root supernatant with FPP
was (+)-germacrene A (7). Additional enzymatic cyclization of this product did not occur. Therefore, we conclude that this (+)-germacrene A synthase activity, present in both wild and cultivated chicory, represents the first dedicated step in the biosynthesis of
bitter compounds in chicory and, more generally, proves the previously
proposed mevalonate-FPP-germacradiene pathway in sesquiterpene lactone biosynthesis (Herz, 1977
Received January 20, 1998;
accepted April 26, 1998.
Abbreviation:
FPP, farnesyl diphosphate.
The authors would like to thank J. de Mik for the gift of
chicory roots, Dr. D.P. Piet for synthesis of germacrene B and
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). Since germacranes also
prefer the chair-chair conformation, Cope rearrangement proceeds easily
(Takeda, 1974). E,E-germacrenes are relatively flexible molecules, but
in the case of a large substituent at C7, the conformation having the substituent at an equatorial position predominates (Takeda,
1974; Piet et al., 1995a). Hence, the two enantiomers of germacrene A
(Fig. 5, 7a and 7b)
will yield two enantiomers of -elemene upon Cope rearrangement (Fig.
5, 10a and 10b, respectively); the diastereomers
10c and 10d will not be formed because the
germacrene conformations 7a
and 7b are
energetically unfavorable. This also explains why the -elemene
diastereomers 10c and 10d have not been found in
nature (Connolly and Hill, 1991).
View larger version (17K):
[in a new window]
Figure 5.
Conformations of the enantiomers of germacrene A
([+]-enantiomer 7a; [ ]-enantiomer 7b), and
their relation to the configuration of the -elemenes formed by Cope
rearrangement.
-selinene [Fig. 3, 8],
-selinene [Fig. 3, 9], selina-4,11-diene [Fig. 3,
11], and ()--elemene [Fig. 5,
10a]), the germacrene A was almost completely rearranged into ()--elemene at an injection port temperature of
250°C (Fig. 6, A and B). This
rearrangement of chicory germacrene A into ()--elemene (and not
into [+]--elemene) was substantiated by co-injection of the
germacrene A with a racemic mixture of -elemene at an injection port
temperature of both 150°C and 250°C (Fig. 6, C and D). The
(+)-enantiomer of -elemene (10b) (König et al.,
1994; Teisseire, 1994) has the same absolute configuration as
()-germacrene A (7b), was determined by Weinheimer et al.
(1970). Considering our experiment where no trace of (+)--elemene was observed and only its counterpart ()--elemene (10a) was detected, we conclude that the germacrene A synthase of
chicory produces exclusively (+)-germacrene A (7a)
(Fig. 7).
View larger version (10K):
[in a new window]
Figure 6.
Determination of the absolute configuration of
germacrene A using GC-MS equipped with a 25-m (0.25-mm i.d.)
heptakis(6-O-TBDMS-2,
3-di-O-methyl)- -cyclodextrin (50% in OV17) chiral
column. Enzymatically (DEAE-purified enzyme) produced germacrene A
(39.66 min) that is stable at an injection port temperature of 150°C
(A) is rearranged into ()--elemene (32.33 min) at an injection
port temperature of 250°C (B). Co-injection of the germacrene A with
a racemic -elemene standard at 150°C (C) and 250°C (D) confirms
the identity of its rearrangement product that co-elutes with
()--elemene and is separated from the (+)-enantiomer of
-elemene (32.15 min). Small amounts of -selinene (8)
(38.52 min), -selinene (9) (38.61 min), and
selina-4,11-diene (11) (38.92 min) were detected during all
measurements.
View larger version (16K):
[in a new window]
Figure 7.
Upon heating, the (+)-germacrene A
(7a) produced in the enzyme assay rearranges toward
( )--elemene (10a) preserving its chiral center. Since
chicory does not produce ()-germacrene A, no (+)--elemene
(10b) was observed. The stereochemical configuration of
(+)-germacrene A (7a) is in accordance with the
stereochemistry of the sesquiterpene lactones in chicory.
80°C, and they served as a stock for all further
experiments.
View this table:
Table I.
Purification of the chicory germacrene A synthase
from 75 mL of a 100,000g supernatant ( 
34 g of root material)
with Red A and Reactive Blue 2. Since Reactive
Green 5 gave the best results and also since it had been used
successfully in the purification of a
trans--farnesene synthase from pine needles (Salin
et al., 1995), we chose to carry out our experiments with it. As shown in Table I, we obtained a recovery slightly above 100% and a 9-fold purification. To ensure a good interaction of the germacrene A
synthase with the matrix, it was important to warm the sample (at
80°C stored DEAE-stock; 1 mL) to room temperature before applying
it to the Reactive Green 5 column. For enzyme stability the fractions
containing enzyme activity required quick desalting into buffer C.
). An additional advantage of this method is that it concentrated
the enzyme activity, which was diluted over 8 mL after the dye-ligand
purification step. Enzyme activity eluted from the Mono-Q column in two
fractions of 0.75 mL at 0.15 M KCl with a recovery of 61%.
A total purification fold of 201 was obtained. If this last
purification step had been carried out in the absence of Tween 20, almost no sesquiterpene cyclase activity would have been detected.
;
Görg et al., 1987).
1
mg1 protein.
and Davis et al. (1996).
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Bohlman and Zdero, 1978; Fischer, 1990; Song et al., 1995). Germacrene B, which would be a likely intermediate from a chemical point of view, appears not to be involved
in the biosynthesis of C8-oxygenated guaianolides in chicory.
and Threlfall and Whitehead (1988)
support this conclusion. Germacrene A is enzymatically oxidized to a
compound having a mass spectrum that is identical to that of
elema-1,3,11(13)-trien-12-ol (Maurer and Grieder, 1977; J.-W. de
Kraker, unpublished results). Most probably, it is the
Cope-rearrangement product of 1(10),4,11(13)-germacratrien-12-ol, indicating that, in chicory, germacrene A is further metabolized into a
12-hydroxylated germacrene.
; Cane, 1990) (Fig. 8).
20°C (Fig. 3)
(Weinheimer et al., 1970; Bowers et al., 1977; Teisseire 1994). Our
experiments showed otherwise. When silica was used during the assay
extraction-filtration procedure, one-half of the germacrene A was
cyclized toward -selinene (8), -selinene (9), and selina-4,11-diene (11). To our knowledge the latter compound has not been reported before in this context, but
originates from the same intermediate carbocation as the other two
selinenes. Using neutral aluminum oxide instead of the slightly acidic
silica effectively minimized the nonenzymatic cyclization. Cope
rearrangement of germacrene A did not occur, not even during incubations at 30°C overnight.
-elemene (10) can be a problem in
GC measurements due to the high temperatures involved. However, reducing the injection port temperature to 150°C greatly diminished Cope rearrangement. If cold on-column injection is applied, no Cope
rearrangement will be observed at all. Rearrangement of germacrene A
and germacrene B during GC-MS measurement was detected by Wichtman and
Stahl-Biskup (1987), whereas the influence of GC injection port
temperature on Cope rearrangement was studied for germacrone by
Reichardt et al. (1988). Nevertheless, high injection port temperatures in combination with enantioselective GC proved to be very
useful in determining the absolute configuration of the germacrene A
formed by the isolated enzyme. The heat-induced Cope rearrangement is
stereospecific and the chiral center at C7 is not involved in
this reaction (Weinheimer et al., 1970; Takeda, 1974; March, 1992)
(Fig. 5). Since only ()--elemene (10a) was obtained, we
can designate our enzymatic product as the (+)-enantiomer of germacrene
A (7a) (Figs. 6 and 7).
-methylene--lactone ring in which the proton
at the C7-position of the sesquiterpenoid
framework is, without exception, -oriented (Bachelor and Ito, 1973;
Seto et al., 1988; Fischer 1990; van Beek et al., 1990).
Therefore, the absolute configuration of (+)-germacrene A corresponds
with its biochemical fate, and the configuration is already determined in the first step of sesquiterpene lactone biosynthesis (Fig. 7).
). In a first step FPP is bound to the
enzyme and dephosphorylated, generating germacrene A. The germacrene A
intermediate is then once more cyclized toward a eudesmane
carbocation whose final destination, either epi-aristolochene or
vetispiradiene, depends upon the particular active site conformation of
the sesquiterpene cyclase involved (Starks et al., 1997).
).
); however, so far it has never been detected because it remains bound to the sesquiterpene cyclase (Cane et
al., 1990; Cane and Tsantrizos, 1996). Nevertheless, various species
such as the liverwort F. tamarisci (W.A. König, unpublished results), caraway (Wichtman and Stahl-Biskup, 1987), the
gorgonian Eunice mammosa (Weinheimer et al., 1970), and the spotted alfalfa aphid (Bowers et al., 1976) contain germacrene A and
should therefore also contain the corresponding germacrene A synthase.
). Consequently, it is rather peculiar that chicory contains an
enzyme that releases germacrene A. Why is FPP not immediately cyclized,
within one enzymatic step, toward a eudesmane or a guaiane by a
(hypothetical) eudesmane synthase or guaiane synthase? In other words,
why is germacrene A released by the chicory sesquiterpene cyclase
instead of being subjected to a second cyclization step? It seems that
in sesquiterpene lactone biosynthesis a different type of reaction
is needed before such a second cyclization can take place.
), as well as the
study of acid-induced cyclizations of germacrene B epoxides (Brown et
al., 1975; Piet et al., 1995b), it has been postulated that this second
cyclization is directed by epoxidations. A germacradienolide
C4-C5-epoxide would be
cyclized to a guaianolide, whereas a germacradienolide
C1-C10-epoxide would be
cyclized to a eudesmanolide (Fischer, 1990; Teisseire, 1994; Song et
al., 1995). Such a cyclization of germacradienolide epoxides toward either a guaianolide or a eudesmanolide, depending on the position of
the epoxide, is presumed to be catalyzed by one and the same germacrane
cyclase that possesses a broad substrate specificity (Piet et al.,
1996).
noticed that all germacranolides and almost all
eudesmanolides of chicory posses a glucosilated hydroxyl function at
C3, whereas the guaianolides lack this function
and have an olefinic C3 atom. An alternative
biopathway (Fig. 9) is proposed in which
this C3-hydroxyl function plays a crucial role. As we have demonstrated for chicory, sesquiterpene lactone biosynthesis starts with the cyclization of FPP to (+)-germacrene A. Through several
oxidative steps (+)-germacrene A might be transformed into a
germacranolide (12), which would be the branching point in
the biosynthesis of guaianolides, eudesmanolides, and germacranolides.
So far, this compound has not been detected in chicory and is probably
quickly subjected to the next biochemical step, just like germacrene A,
that has not been detected in vivo either. Cyclization of 12 by a germacrane cyclase would start with the protonation of the
C3-hydroxyl group, which is then released as a
water molecule. The carbocation thus formed leads to guaianolides via
1,5-cyclization. Glucosylation of the C3-hydroxyl
function would prevent this type of cyclization; it leaves the
germacranolides as such or leads to the germacrane cyclase mediated
cyclization toward eudesmanolides.
View larger version (14K):
[in a new window]
Figure 9.
Proposed pathway for the biosynthesis of
sesquiterpene lactones in chicory using the (+)-germacrene A synthase
as a starting point. It is not known at which stages the oxidative
steps take place.
1
Both of these authors contributed equally
to this manuscript; e-mail M.C.R.F.: maurice.franssen{at}bio.oc.wau.nl;
fax 31-317-48-4914; e-mail H.J.B.: H.J.Bouwmeester{at}ab.dlo.nl;
fax 31
FOOTNOTES
317423110.
*
Corresponding author.
ABBREVIATIONS
ACKNOWLEDGMENTS
-elemene, Dr. M.A. Posthumus and J.A.R. Davies for their help with
the GC-MS analyses, and M.C.J.M. Konings for technical assistance. We
also thank the Koninklijke Landbouwkundige Vereniging for their support during the first stages of the work presented.
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-selinene cyclase from Citronella mitis fruits.
Plant Sci
84:
129-136
-cadinene synthase, a sesquiterpene cyclase from bacteria inoculated cotton foliar tissue.
Phytochemistry
41:
1047-1055
[CrossRef][Web of Science][Medline]146-ol and (+)-hedycaryol by Cichorium intybus.
Tetrahedron
51:
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[CrossRef]-farnesene synthase from maritime pine (Pinus pinaster Ait.) needles.
J Plant Physiol
146:
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)-germacrene-A from the gorgonian, Eunicea mammosa: chemistry of coelenterates XVIII.
Tetrahedron Lett
7:
497-500
Copyright Clearance Center: 0032-0889/98/117//12
© 1998 American Society of Plant Physiologists
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) and 2-fold-diluted Mono-Q eluent (
). B,
Michaelis-Menten curve featuring a Km of 6.6 µM and a Vmax of 66.8 pmol
h
, 
),
Mes (
, 
), Mopso (
), and Tris-HCl (
). Also shown
is the pH curve for Mono-Q-purified enzyme in the presence of Tween 20 (×). D, Determination of the Mr by
calibrated gel filtration. The column is calibrated by measuring the
elution volume (