Biosynthesis of the hyperforin skeleton in Hypericum calycinum cell cultures

Article abstract of DOI:10.1016/j.phytochem.2004.11.003

Hyperforin is an important antidepressant constituent of Hypericum perforatum (St. John's wort). Cell cultures of the related species H. calycinum were found to contain the homologue adhyperforin and to a low extent hyperforin, when grown in BDS medium in

Full text of DOI:10.1016/j.phytochem.2004.11.003

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 139–145
www.elsevier.com/locate/phytochem
Biosynthesis of the hyperforin skeleton in Hypericum calycinum
cell cultures
a
Petra Klingauf , Till Beuerle , Annett Mellenthin , Safaa A.M. El-Moghazy
a
b
a,1
,
a
Zakia Boubakir , Ludger Beerhues
a,
*
a
Institut fu¨r Pharmazeutische Biologie, Technische Universita¨t Braunschweig, Mendelssohnstraße 1, D-38106 Braunschweig, Germany
Institut fu¨r Lebensmittelwissenschaft und Lebensmittelchemie, Universita¨t Bonn, Endenicher Allee 11-13, D-53115 Bonn, Germany
b
Received 3 September 2004; received in revised form 10 November 2004
Available online 18 December 2004
Abstract
Hyperforin is an important antidepressant constituent of Hypericum perforatum (St. JohnÕs wort). Cell cultures of the related
species H. calycinum were found to contain the homologue adhyperforin and to a low extent hyperforin, when grown in BDS med-
ium in the dark. Adhyperforin formation paralleled cell culture growth. Cell-free extracts from the cell cultures contained isobutyr-
ophenone synthase activity catalyzing the condensation of isobutyryl-CoA with three molecules of malonyl-CoA to give
phlorisobutyrophenone, i.e. the hyperforin skeleton. The formation of the hyperforins during cell culture growth was preceded
by an increase in isobutyrophenone synthase activity. The cell cultures also contained benzophenone synthase and chalcone synthase
activities which are involved in xanthone and flavonoid biosyntheses, respectively. The three type III polyketide synthases were sep-
arated by anion exchange chromatography.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Hypericum calycinum; Hyperforins; Isobutyrophenone synthase; Benzophenone synthase; Chalcone synthase; Cell cultures
1. Introduction
contain appreciable quantities of xanthones which also
exhibit antidepressant properties (Muruganandam
et al., 2000).
Hyperforin (Fig. 1) is a polyprenylated acylphloro-
glucinol derivative and inhibits non-selectively the reup-
take of a number of neurotransmitters (Muller, 2003).
¨
The homologue adhyperforin is a minor constituent of
H. perforatum and exhibits similar pharmacological
properties (Maisenbacher and Kovar, 1992; Jensen
et al., 2001). In this study, we report the occurrence of
hyperforin and adhyperforin in cell cultures of H. cal-
ycinum and the detection of three type III polyketide
synthases (PKS), one of which participates in hyperforin
biosynthesis.
Alcoholic extracts from the flowering upper parts of
Hypericum perforatum L. (St. JohnÕs wort) are widely
used for the treatment of mild to moderate depression
(Butterweck, 2003; Muller, 2003). Their antidepressant
¨
activity is attributed to hyperforins, flavonoids and hyp-
ericins (Butterweck, 2003). Indian H. perforatum plants
Abbreviations: BPS, benzophenone synthase; BUS, isobutyrophe-
none synthase; CHS, chalcone synthase; DEAE, diethylaminoethyl;
DTT, dithiothreitol; PKS, polyketide synthase.
*
Corresponding author. Tel.: +49 531 3915689; fax: +49 531
3918104.
Type III PKS generate a diverse array of secondary
metabolites by condensing multiple acetyl units derived
from malonyl-CoA to specific starter molecules (Jez
E-mail address: l.beerhues@tu-bs.de (L. Beerhues).
1
Permanent address: Department of Pharmacognosy, Faculty of
Pharmacy, Assiut University, Assiut 71526, Egypt.
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.11.003

140
P. Klingauf et al. / Phytochemistry 66 (2005) 139–145
dry weight
adhyperforin
0.35
0.3
12
10
8
0.25
0.2
HO
O
6
R
0.15
0.1
4
O
O
2
0.05
0
0
0
2
4
6
8
10
12
days
Fig. 2. Growth and adhyperforin formation of H. calycinum cell
cultures. Bars represent means and standard deviations from three
determinations.
hyperforin R = H
adhyperforin R = CH3
(Fig. 2). The formation of adhyperforin paralleled cell
culture growth, i.e. accumulation increased linearly from
day 2 to day 6, with the maximum yield being reached at
day 8. The adhyperforin content was 0.03% of dry
weight. The hyperforin content was approx. one tenth
of the adhyperforin concentration. No hyperforins were
detected in the cell culture medium.
Fig. 1. Chemical structures of hyperforins.
et al., 2002). The homodimeric enzymes orchestrate a
series of acyltransferase, decarboxylation, condensation,
cyclization, and aromatization reactions at two func-
tionally independent active sites. The structure of the ac-
tive site dictates starter substrate preference, polyketide
chain-length, and regio-specificity of polyketide cycliza-
tion. These strategies have increased considerably the
functional diversity of type III PKS.
2.3. Detection of isobutyrophenone synthase (BUS)
activity
Incubation of desalted cell-free extracts from
H. calycinum cell cultures with isobutyryl-CoA and
malonyl-CoA resulted in the formation of phlorisobu-
tyrophenone (Fig. 3). The enzymatic product was iden-
tified by HPLC and GC–MS in comparison with a
sample of chemically synthesized reference compound
(Fig. 4). Besides co-chromatography, the mass spectrum
of the acetylated enzymatic product was identical with
that of the acetylated synthetic product. No formation
of the enzymatic product occurred when incubation
assays contained heat-denatured protein extract.
2. Results
2.1. Detection of hyperforins
Cell cultures of H. calycinum formed adhyperforin
and to a low extent hyperforin, when grown in BDS
medium in the dark. Light repressed the formation of
hyperforins, as previously observed with xanthone pro-
duction in cultured cells of H. androsaemum (Schmidt
et al., 2000). The identity of the two hyperforins was
shown by co-chromatography (HPLC) with samples of
authentic reference compounds and HPLC–MS. The
mass spectra of the constituents were consistent with
those of the authentic reference compounds and agreed
with published data (Maisenbacher and Kovar, 1992;
Erdelmeier, 1998). The level of hyperforins in cultured
cells was not affected by addition of methyl jasmonate
and jasmonoyl-isoleucine. Both elicitors also failed to
induce hyperforin formation in cell cultures of H. andro-
saemum, H. gnidioides, and H. perforatum.
2.4. Detection of benzophenone synthase (BPS) and
chalcone synthase (CHS) activities
Enzymatic conversions were also observed when iso-
butyryl-CoA as starter substrate was replaced by ben-
zoyl-CoA and 4-coumaroyl-CoA (Fig. 3). The
products formed were identified as phlorbenzophenone
and naringenin, respectively, by co-chromatography
with samples of authentic reference compounds.
2.5. Separation of three PKS by anion exchange
chromatography
2.2. Characterization of H. calycinum cell cultures
After a 2-day-lag phase, the dry weight of cultured
cells increased from day 2 to day 4 before the cell cul-
tures entered the deceleration and stationary phases
Cell-free extracts were subjected to ammonium sul-
phate precipitation. The protein fraction precipitating
between 55% and 70% saturation was chromatographed

P. Klingauf et al. / Phytochemistry 66 (2005) 139–145
141
Isobutyrophenone synthase:
O
SCoA
O
O
HO
OH
O
3-Malonyl-CoA
CoAS
O
O
OH
isobutyryl-CoA
phlorisobutyrophenone
Benzophenone synthase:
O
SCoA
HO
OH
O
O
3-Malonyl-CoA
CoAS
O
O
O
O
H
benzoyl-CoA
phlorbenzophenone
Chalcone synthase:
OH
OH
OH
O
SCoA
O
HO
OH
O
O
3-Malonyl-CoA
CoAS
O
O
OH
4-coumaroyl-CoA
naringenin chalcone
Fig. 3. Reactions catalyzed by three type III PKS from H. calycinum cell cultures.
on a DEAE-anion exchange column, resulting in the
separation of three PKS activities (Fig. 5). In the order
of elution, the enzymes preferred benzoyl-CoA, isobuty-
ryl-CoA and 4-coumaroyl-CoA as starter substrates and
converted the respective two other starter substrates less
efficiently. Thus, the three PKS resolved were BPS, BUS
and CHS, respectively.
plants of H. calycinum, although several dearomatized
polyprenylated acylphloroglucinols have been isolated
from this species (Decosterd et al., 1998; Gronquist
et al., 2001). Where the lipophilic compounds accumu-
late in differentiated plants and cultured cells of Hyper-
icum species is still open.
Three type III PKS were detected in cell-free extracts
from H. calycinum cell cultures and separated by anion
exchange chromatography. One of them, BUS, is
responsible for the formation of the hyperforin skeleton.
It catalyzes the condensation of one molecule of isobu-
tyryl-CoA with three molecules of malonyl-CoA to give
phlorisobutyrophenone. As expected, an increase in
BUS activity preceded formation of hyperforins. While
isobutyryl-CoA is the starter substrate for hyperforin,
adhyperforin is derived from 2-methylbutyryl-CoA.
The latter substrate appears to be the preferred starter
molecule in cell cultures of H. calycinum which contain
mainly adhyperforin.
An enzyme which functionally resembles BUS par-
ticipates in the biosynthesis of bitter acids in glandular
hairs of hop cones. This enzyme was purified (Paniego
et al., 1999) and its gene was isolated (Okada and Ito,
2001). In both species, Humulus lupulus and H. calyci-
num, the PKS products undergo stepwise prenylation
to give bitter acids and hyperforins, respectively.
Besides BUS, H. calycinum cell cultures contained BPS
and CHS which form the carbon skeletons of xan- thones
and flavonoids, respectively. Both classes of compounds
have previously been found in intact H. calycinum plants
(Decosterd et al., 1998; Gronquist et al., 2001). While
CHS is the best-known member of the superfamily of
2.6. Changes in the PKS activities during cell culture
growth
BUS activity rapidly increased after cell culture trans-
fer into new growth medium (Fig. 6). Thereafter, the en-
zyme activity was high between day 1 and day 4. In
contrast, CHS activity increased slowly and reached its
maximum at days 4–5. BPS activity appeared to be high
at day 3. Its starter substrate benzoyl-CoA was also effi-
ciently converted by BUS but this enzyme activity exhib-
ited the plateau between day 1 and day 4. The maximum
specific activities of BUS and CHS were 0.85 and
1.4 lkat/kg. The specific activity with benzoyl-CoA as
starter molecule increased to 2.5 lkat/kg.
3. Discussion
Cell cultures of H. calycinum were found to be a valu-
able in vitro system for studying hyperforin biosynthe-
sis. In contrast to intact plants of H. perforatum which
accumulate hyperforin, cell cultures of H. calycinum
contained mainly the homologue adhyperforin. So far,
hyperforins have not been detected in differentiated

142
P. Klingauf et al. / Phytochemistry 66 (2005) 139–145
type III PKS (Schro¨der, 1999), BPS has been cloned only
recently from cell cultures of H. androsaemum (Liu et al.,
2003). BPS preferred benzoyl-CoA as starter substrate
and discriminated 4-coumaroyl-CoA which, however,
was the most efficient starter molecule for CHS. The
products resulting from these two PKS reactions are
phlorbenzophenone and naringenin chalcone. These
intermediates can undergo intramolecular cyclization to
give xanthones and flavonoids, respectively.
Another class of active Hypericum constituents are
hypericins. These naphthodianthrones result from
dimerization of emodin anthrone (Bais et al., 2003).
The biosynthesis of these octaketide-derived monomers
might be catalyzed by a type III PKS. Recently, a novel
CHS-like protein from Rheum palmatum was found to
form an aromatic heptaketide (Abe et al., 2004). In con-
clusion, polyketide metabolism plays a central role in
the biosynthesis of the active Hypericum constituents.
4. Experimental
4.1. Chemicals
Hyperforin and adhyperforin were a kind gift from
Dr. Erdelmeier (Dr. Willmar Schwabe, Karlsruhe, Ger-
many). In addition, hyperforin was purchased from
HWI Analytik (Rheinzabern, Germany). Methyl jasmo-
nate was obtained from Serva (Heidelberg, Germany).
Jasmonoyl-isoleucine was kindly provided by Prof. W.
Boland (MPI for Chemical Ecology, Jena, Germany).
Benzoyl-CoA, isobutyryl-CoA and malonyl-CoA were
obtained from Sigma (Deisenhofen, Germany). 4-Cou-
maroyl-CoA was synthesized as reported earlier (Abd
El-Mawla and Beerhues, 2002). Phlorbenzophenone
and naringenin were purchased from ICN (Mecken-
Fig. 4. GC–MS analysis of BUS assays. The enzymatic product and
the reference compound were acetylated. Phthalate, hexadecanoic acid,
hexadecanoic acid ethyl ester and an unknown aliphatic compound
were also detected in control assays containing heat-denatured cell-free
extract.
Fig. 5. Separation by DEAE-anion exchange chromatography of BPS, BUS and CHS from H. calycinum cell cultures. Data are mean values of two
independent experiments.

P. Klingauf et al. / Phytochemistry 66 (2005) 139–145
143
isobutyrophenone synthase
4.3. Extraction of hyperforins
1
0.8
0.6
0.4
0.2
0
Cultured cells (3 g) were collected by suction filtration
and homogenized in a mortar with 5 ml CHCl3 or
CH2Cl2 for 5 min. After centrifugation at 10,000g for
10 min, the organic phase was evaporated to dryness
in vacuo and the residue dissolved in 500 ll MeOH.
The procedure was carried out at 0–4 ꢁC.
0
1
2
3
4
5
6
7
4.4. HPLC
1.5
1.25
1
chalcone synthase
Hyperforins were separated on a Nucleosil C18 100-5
column (25 · 0.4 cm; Macherey-Nagel, Duren, Ger-
¨
0.75
0.5
0.25
0
many) at a flow rate of 1 ml/min. The solvent was
MeOH (85%) and the detection wavelength 275 nm.
The Rt value of hyperforin was 6.0, that of adhyperforin
6.7. Standard solutions of authentic compounds were
used for quantification.
0
1
2
3
4
5
6
7
3
2.5
2
Enzymatic products were analyzed using the solvents
H2O (A) and MeOH (B) and the following gradient:
40% B for 3 min, 40–70% B in 14 min, then isocratic
at 70% B. The detection wavelengths were 285 nm for
naringenin, 288 nm for phlorisobutyrophenone, and
306 nm for phlorbenzophenone.
benzoyl-CoA as starter substrate
1.5
1
4.5. Spectrometric methods
0.5
0
1
2
3
4
5
6
7
days
4.5.1. LC–MS
Analyses were performed on a Beckmann Gold Nou-
veau HPLC system with gradient pump 126 (Beckmann
Coulter, Unterschleißheim, Germany) equipped with a
Nucleosil C18 100-5 column (25 · 0.4 cm). MeOH
(85%) served as a solvent at a flow rate of 1 ml/min.
Mass detection and fragmentation experiments were
performed on a Finnigan MAT LCQ mass spectrometer
(Thermo-Quest, Egelsbach, Germany) equipped with an
ESI source and an ion trap mass analyzer which was
controlled by the LCQ Navigator software. The LCQ
was operated in the negative ion mode under the follow-
ing conditions: source voltage 5 kV, capillary voltage ꢀ5
V, and capillary temperature 280 ꢁC.
Fig. 6. Changes in the specific activities of BUS and CHS during cell
culture growth. Benzoyl-CoA as starter substrate was efficiently
converted by both BPS and BUS. Bars represent means and standard
deviations from three determinations.
heim, Germany) and Sigma (Taufkirchen, Germany),
respectively.
4.2. Cell cultures and elicitation
Callus of H. calycinum was initiated from young
shoots collected in the Botanical Garden of the Univer-
sity of Bonn, Germany. Surface-sterilized stem segments
were placed on LS (Linsmaier and Skoog, 1965), MS
(Murashige and Skoog, 1962), 4· (Peters et al., 1998)
and BDS media (Gamborg and Eveleigh, 1968). 4·
and BDS are modified B5 media. Callus aliquots were
transferred to the respective liquid media and the result-
ing cell cultures (50 ml) were shaken in Erlenmeyer
flasks (300 ml) at 100 rpm and 25 ꢁC either in the dark
or under continuous light. Cell cultures were propagated
by weekly transfer of aliquots (4 g) of the fine suspen-
sions into fresh growth medium.
4.5.2. GC–MS
Acetylation of products and subsequent analysis were
carried out as described previously (Liu et al., 2004).
4.6. Synthesis of phlorisobutyrophenone (2-methyl-
1-(2,4,6-trihydroxyphenyl)-1-propanone)
The reaction was conducted as described by van
Klink et al. (1999) starting with 2 g (12 mmol) of dry
phloroglucinol (120 ꢁC for 12 h). This was added to
a solution of anhydrous AlCl3 (4 g; 120 ꢁC for 12 h)
in phosphorous oxychloride (15 ml) under N2 at 0
ꢁC. Twelve mmol of 2-methylpropanoic acid (1.1 g)
were added dropwise and the resulting solution was
Four-day-old cell cultures were treated with either
methyl jasmonate or jasmonoyl-isoleucine (100 lmol/l
each). After 48 h, cultured cells were extracted and
hyperforins quantitatively analyzed.

144
P. Klingauf et al. / Phytochemistry 66 (2005) 139–145
stirred for 8 h at 0 ꢁC under N2, then for 48 h at ca.
6 ꢁC. The reaction mixture was poured on crushed
ice (ca. 100 ml) and the resulting mixture extracted
three times with 100 ml of diethyl ether. The organic
phases were combined and washed with saturated
NaHCO3 solution (2 · 250 ml) and dried over anhy-
drous Na2SO4. The organic solvent was removed and
the crude reaction product chromatographed on a sil-
ica gel 60 column (2.5 · 40 cm) using the solvent
diethyl ether:pentane (70:30). The NMR spectrum of
the purified compound was in agreement with pub-
lished data (Amer et al., 1983). For GC–MS, the com-
pound was acetylated (Section 4.5).
4.9. Anion exchange chromatography
Crude extract from 2-day-old cultured cells (20 g) was
subjected to ammonium sulphate precipitation. The pro-
tein fraction precipitating between 55% and 70% satura-
tion was passed through a PD-10 column and loaded
onto a DEAE-anion exchange column (HiTrap, 1 ml)
connected to a BioLogic System (Bio-Rad, Munchen,
¨
Germany). Both columns were purchased from Amer-
sham Biosciences (Freiburg, Germany) and equilibrated
with 20 mM Tris-HCL, pH 7.4 containing 1 mM DTT.
After removal of unbound proteins by washing with
three gel volumes of the same buffer, bound proteins
were eluted with a linear gradient from 0 to 500 mM
KCl at a flow rate of 2 ml/min. Fractions of 1 ml were
collected.
4.6.1. 2-Methyl-1-(2,4,6-trihydroxyphenyl)-
1-propanone
Rf (CH2Cl2:EtOAc:HOAc 79:20:1, silica gel) 0.38,
(CH2Cl2:EtOAc:HOAc 69:30:1, silica gel) 0.84; RI
(ZB1) 1930; 1H NMR (400 MHz, acetone-d6) d 1.13
(6H, d, 2xCH3, J = 7Hz), 3.97 (1H, sept., (CH3)2CH),
5.93 (2 H, s, Carom.H-3 and H-5), 9.14 (1H, s, OH),
11.7 (2H, s, OH). EIMS, 70eV, m/z (rel. int.): 196 (22,
[M]+), 153 (100, [M ꢀ C3H7]+), 154 (7), 69 (6), 43 (4),
41 (4), 197 (2), 111 (2), 67 (2), 55 (2).
Acknowledgements
We thank Dr. C. Erdelmeier (Dr. Willmar Schwabe
GmbH & Co., Karlsruhe, Germany) and Prof. W. Bo-
¨
land (Max-Planck-Institut fur Chemische Okologie,
¨
Jena, Germany) for samples of hyperforin/adhyperforin
and jasmonoyl-isoleucine, respectively. This work was
supported by a Sokrates scholarship from the European
Union (to P. Klingauf), a research fellowship from the
Egyptian government (to S.A.M. El-Moghazy), a schol-
arship from the Libyan government (to Z. Boubakir),
and a grant from the Deutsche Forschungsgemeinschaft
(to L. Beerhues).
4.6.2. 2-Methyl-1-(2,4,6-tri-O-acetylphenyl)-
1-propanone
RI (ZB1) 2031; EIMS, 70eV, m/z (rel. int.): 322 (0.4,
[M]+), 153 (100, [M ꢀ C3H7 ꢀ 3(H2C@C@O)]+), 195
(84, [M ꢀ C3H7 ꢀ 2(H2C@C@O)]+), 237 (59, [M ꢀ
C3H7 ꢀ (H2C@C@O)]+), 279 (53, [M ꢀ C3H7]+), 43 (40,
H3C2O), 238 (19), 280 (15), 196 (11), 152 (10), 154 (7).
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