The first prenylation step in hyperforin biosynthesis

Article abstract of DOI:10.1016/j.phytochem.2004.10.020

Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum

Full text of DOI:10.1016/j.phytochem.2004.10.020

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 51–57
www.elsevier.com/locate/phytochem
The first prenylation step in hyperforin biosynthesis
1
Zakia Boubakir, Till Beuerle, Benye Liu , Ludger Beerhues
*
Institut fuer Pharmazeutische Biologie, Technische Universitaet Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig, Germany
Received 4 June 2004; received in revised form 26 October 2004
Available online 25 November 2004
Abstract
Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include
hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum (St.
JohnÕs wort). Cell cultures of the related species Hypericum calycinum were found to contain a prenyltransferase activity which is
likely to catalyze the first prenylation step in hyperforin biosynthesis. The enzyme was soluble and dependent on a divalent cation,
with Fe2+ leading to maximum activity (Km = 3.8 mM). The preferred prenyl donor was DMAPP (Km = 0.46 mM) and the preferred
prenyl acceptor was phlorisobutyrophenone (Km = 0.52 mM). A broad pH optimum from 6.5 to 8.5 and a temperature optimum
from 35 to 40 ꢀC were observed. The formation of hyperforins in H. calycinum cell cultures was preceded by an increase in dimeth-
ylallyltransferase activity, with the maximum specific activity being 3.6 lkat/kg protein.
ꢁ 2004 Elsevier Ltd. All rights reserved.
Keywords: Hypericum calycinum cell cultures; Hyperforin biosynthesis; Prenylation; Phlorisobutyrophenone dimethylallyltransferase
1. Introduction
ral products, such as coumarins and flavonoids. Cycli-
zations can also result from formation of carbon–
carbon bonds, giving complex molecules with caged
skeletons. These bridged polycyclic systems are fre-
quently found in phloroglucinol derivatives, such as
prenylated benzophenones (Hu and Sim, 2000) and
hyperforins.
Hyperforin (Fig. 1) is a secondary metabolite with
interesting pharmacological properties. It contributes
to the antidepressant activity of St. JohnÕs wort
(Hypericum perforatum) extracts by inhibiting non-se-
lectively the synaptosomal reuptake of a number of
Many natural products contain terpenoid residues.
Carbon skeletons derived for example from the shiki-
mate and polyketide pathways can be modified by
attaching isoprenoid chains. A particularly common
terpenoid group is the dimethylallyl substituent. This
residue is able to cyclize with an ortho hydroxyl group,
leading to the formation of either five-membered furan
or six-membered pyran heterocycles (Stanjek et al.,
1999). These rings are commonly encountered in natu-
neurotransmitters (Muller, 2003). The underlying mode
¨
Abbreviations: DMAPP, dimethylallyl diphosphate; IPP, isopente-
nyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphos-
phate; GGPP, geranylgeranyl diphosphate.
of action is unique to hyperforin which does not di-
rectly interact with the transmitter transporters but ele-
vates the intracellular sodium concentration, thereby
inhibiting the gradient-driven neurotransmitter uptake.
However, hyperforin also binds to the pregnane X
receptor which regulates the expression of CYP 3A4
(Moore et al., 2000; Cantoni et al., 2003). The
*
Corresponding author. Tel.: +49 531 391 5689; fax: +49 531 391
8104.
E-mail address: l.beerhues@tu-bs.de (L. Beerhues).
Present address: Key Laboratory of Photosynthesis and Environ-
1
mental Molecular Physiology, Institute of Botany, The Chinese
Academy of Sciences, Beijing 100093, China.
0031-9422/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.10.020

52
Z. Boubakir et al. / Phytochemistry 66 (2005) 51–57
HO
OH
O
HO
O
O
OH
O
HO
DMAPP
OH
O
OH
hyperforin
phlorisobutyrophenone
dimethylallyl-
phlorisobutyrophenone
Fig. 1. Prenylation of the hyperforin nucleus by phlorisobutyrophenone dimethylallyltransferase.
compound thus contributes to both the therapeutic and
the side effects of St. JohnÕs wort preparations. Hyper-
forin also exhibits antibiotic properties (Schempp et al.,
1999) and inhibits in vivo the growth of human and rat
tumour cell lines by induction of apoptosis (Schempp
et al., 2002).
Little is known about the biosynthesis of hyperforin.
Recently, the formation of hyperforin and its homo-
logue adhyperforin was observed in cell cultures of
Hypericum calycinum which are thus a valuable in vitro
system for studying hyperforin biosynthesis (Klingauf
et al., 2005). The cultured cells contained, besides chal-
cone synthase and benzophenone synthase, isobutyro-
phenone synthase which catalyzes the sequential
condensation of one molecule of isobutyryl-CoA with
three molecules of malonyl-CoA to form the hyperforin
nucleus (Klingauf et al., 2005). This aromatic intermedi-
ate undergoes a series of prenylation reactions. Here we
report the first prenylation step.
2.2. Substrate specificity and kinetic data
The transferase used DMAPP as prenyl donor (Table
1). Product formation also occurred in the presence of
IPP, which, however, was attributed to IPP isomerase
activity in the crude extract. No enzyme activity was ob-
served with GPP and FPP as prenyl donors. The pre-
ferred prenyl acceptor was phlorisobutyrophenone
(Table 1). High transferase activity was also found with
phloracetophenone. The product resulting from this
acceptor was identified as dimethylallylphloracetophe-
none by HPLC and GC–MS in comparison with a sam-
ple of chemically synthesized reference compound. No
enzyme activity occurred with phloroglucinol as prenyl
acceptor. Prenylation of phlorisobutyrophenone fol-
lowed Michaelis–Menten kinetics. The Km values were
0.52 mM for the acceptor and 0.46 mM for DMAPP,
as calculated from Lineweaver–Burk plots (Fig. 3).
The formation of hyperforins in H. calycinum cell cul-
tures occurred between day 2 and 6 (Klingauf et al.,
2005) and was preceded by an increase in dimethylallyl-
transferase activity (Fig. 4). The maximum specific en-
zyme activity was 3.6 lkat/kg protein.
2. Results
2.1. Detection of prenyltransferase activity
2.3. Metal ion dependence, solubility and pH and
temperature optima
Phlorisobutyrophenone and DMAPP were chemi-
cally synthesized and incubated with cell-free extracts
from 3-day-old H. calycinum cell cultures in the pres-
ence of a divalent cation. These incubations resulted
in the formation of an enzymatic product, as demon-
strated by HPLC and GC analyses (Fig. 2). No prenyl-
transferase activity could be detected when boiled
protein extract was used and either phlorisobutyrophe-
none or DMAPP was omitted. The enzymatic product
was extracted from large-scale incubations, purified by
TLC and analyzed by GC–MS. In comparison with a
sample of chemically synthesized reference compound,
it was identified as dimethylallylphlorisobutyrophe-
none. The synthetic compound and the enzymatic
product exhibited identical retention times and mass
spectra. This was also true for their acetylated deriva-
tives (Fig. 2).
Dimethylallyltransferase activity was strictly depend-
ent on a divalent cation, with Fe2+ being the most effi-
cient cofactor (Fig. 5). This finding was not due to the
reducing properties of Fe2+ because experiments in the
presence and in the absence of 10 mM ascorbic acid gave
the same result. The Km for Fe2+ was 3.8 mM (Fig. 3).
The prenyltransferase showed a broad pH optimum
from 6.5 to 8.5, which has previously been observed with
other prenyltransferases (Yamamoto et al., 1997). Un-
der the assay conditions described, optimal product for-
mation was observed at 35–40 ꢀC. The reaction was
linear with time up to 30 min and with the protein
amount up to 80 lg in the assay. After ultracentrifuga-
tion, about 80% of the enzyme activity was found in the
supernatant, indicating a soluble dimethylallyltrans-
ferase (Table 2).

Z. Boubakir et al. / Phytochemistry 66 (2005) 51–57
53
Fig. 2. GC–MS analysis of prenyltransferase assays. The control assay contained heat-denatured protein. The enzymatic product and the synthetic
reference compound were acetylated.
3. Discussion
and Zenk, 1998), hop bitter acids (Zuurbier et al.,
1998), and shikonin (Yazaki et al., 2002). The shiko-
nin-related enzyme was the first prenyltransferase to
be cloned from a plant source, namely cell cultures
of Lithospermum erythrorhizon. The enzyme catalyzes
the transfer of a geranyl residue to 4-hydroxybenzoic
acid.
Prenylation reactions contribute considerably to
the diversification of secondary metabolites. Prenyl-
transferases participate in the biosynthesis of
flavonoids (Yamamoto et al., 2000), coumarins
(Hamerski et al., 1990), cannabinoids (Fellermeier

54
Z. Boubakir et al. / Phytochemistry 66 (2005) 51–57
Table 1
Substrate specificity of phlorisobutyrophenone dimethylallyltransferase from H. calycinum cell cultures
Prenyl donor
Rel. enzyme activity (%)
Prenyl acceptor
Rel. enzyme activity (%)
Dimethylallyl diphosphate (DMAPP)
Isopentenyl diphosphate (IPP)
Geranyl diphosphate (GPP)
100a
30
<0.3b
<0.3b
<0.3b
Phlorisobutyrophenone
Phloracetophenone
Phloroglucinol
100a
80
<0.3b
Farnesyl diphosphate (FPP)
Geranylgeranyl diphosphate (GGPP)
a
Specific activity: 3.6 lkat/kg.
Detection limit: 1 ng.
b
prenyltransferase
adhyperforin
1,2
0,8
0,4
0
4
3.5
3
0.35
0.3
0.25
0.2
2.5
2
0.15
0.1
1.5
1
-2
0
2
4
6
0.05
0
1/[DMAPP] (mM-1)
0.5
0
1
2
3
4
5
6
7
8
9
10
days
1,2
0,8
0,4
0
Fig. 4. Adhyperforin formation (data from Klingauf et al., 2005) and
changes in prenyltransferase activity during cell culture growth. Values
are means of two experiments.
4
3,5
3
-4
0
4
8
12
1/[phlorisobutyrophenone] (mM-1)
2,5
2
1,2
1,5
1
0,5
0
0,8
0,4
0
-
Fe2+
Mg2+
Zn2+
Cu2+
Ca2+
Mn2+
Co2+
divalent cations
Fig. 5. Effect of divalent cations on dimethylallyltransferase activity.
Bars represent means and standard deviations from three
determinations.
-0,6
0
0.6
1.2
1.8
1/[Fe2+] (mM-1)
Fig. 3. Dependence of dimethylallyltransferase activity on the con-
centrations of phlorisobutyrophenone, DMAPP, and Fe2+
binding site [(N/D)DXXD] (Pojer et al., 2003). This
binding motif is also present in the chain-elongating
trans-prenyltransferases, such as geranyl diphosphate
synthase, farnesyl diphosphate synthase, and geranyl-
geranyl diphosphate synthase (Wang and Ohnuma,
1999). The cloned geranyltransferase from L. erythrorh-
izon also belongs to this group (Yazaki et al., 2002).
Another class of aromatic prenyltransferases includes
soluble enzymes which, in addition, lack the characteris-
tic prenyl diphosphate binding site and thus the absolute
requirement for a divalent cation. Examples are the
microbial enzymes 4-dimethylallyltryptophan synthase
and CloQ which are involved in ergot alkaloid and
clorobiocin biosyntheses, respectively (Tsai et al., 1995;
Pojer et al., 2003).
.
In this study, we detected and characterized the
dimethylallyltransferase which is likely to catalyze the
first prenylation step in hyperforin biosynthesis. How-
ever, it cannot yet be fully excluded that the enzyme
might be related to the formation of other prenylated
secondary metabolites, such as flavonoids and
benzophenones. The enzyme is a so-called aromatic pre-
nyltransferase which catalyzes the formation of a car-
bon–carbon bond between an aromatic nucleus and a
prenyl group. The majority of the aromatic prenyl-
transferases are integral membrane proteins and their
catalytic cavities include a typical prenyl disphophate

Z. Boubakir et al. / Phytochemistry 66 (2005) 51–57
55
Table 2
Prenyltransferase activity in cell fractions after ultracentrifugation
Cell fraction
Protein (mg)
Total activity
pkat
Specific activity (lkat/kg)
%
Cell-free extract
Supernatant (100,000g)
Pellet (100,000g)
5.5
4.9
0.5
19.6
15.7
0.07
100
3.6
3.2
0.1
80.1
0.4
The dimethylallyltransferase from cultured H. calyci-
num cells differs from the members of the above-men-
tioned classes of prenyltransferases in that it is soluble
and its activity dependent on a divalent cation. It is
the first plant prenyltransferase to prefer Fe2+. Other en-
zymes are most active with Mg2+ or Mn2+ (Hamerski
et al., 1990; Laflamme et al., 1993; Zuurbier et al.,
1998). Prenyltransferases with similar properties, i.e. sol-
uble and ion-dependent, are involved in the biosynthesis
of bitter acids in hop (Humulus lupulus; Zuurbier et al.,
1998) and cannabinoids in hemp (Cannabis sativa; Fel-
lermeier and Zenk, 1998). In glandular hairs of hop
cones, the enzyme activity catalyzes the stepwise preny-
lation of acylphloroglucinols to yield deoxyhumulone
and deoxycohumulone which are the direct precursors
of the hop bitter acids. In leaves of Indian hemp, the en-
zyme catalyzes the alkylation of olivetolic acid with
GPP. It will be interesting to study the evolutionary
relationship between the dimethylallyltransferase from
H. calycinum cell cultures and the enzymes of the two
other classes of prenyltransferases.
reduced pressure. Due to instability in silica gel, the prod-
uct was chromatographed on Sephadex LH-20 (2.5 · 40
cm; Amersham Biosciences, Freiburg, Germany) using
methanol (70%) as solvent. Further purification was
achieved by semi-preparative HPLC (0.8 · 25 cm; Euro-
sorb-100 C18) using water (A) and methanol (B) for the
following gradient: 50% B for 2 min, 50–80% B in 30
min, 80–100% B in 5 min. The flow rate was 3 ml/min
and the detection wavelength 290 nm. The spectroscopic
properties of the product agreed with the literature
(Kuhnke and Bohlmann, 1985; Fung et al., 1994). For
acetylation, the product was dissolved in 10 ll dry pyri-
dine and 10 ll acetic acid anhydride. After 1 h at room
temperature, the reaction was quenched by addition of
500 ll methanol and an aliquot was analyzed by GC–MS.
3-(3-Methyl-2-butenyl)-2,4,6-trihydroxyisobutyrophe-
none: Rf (silica gel, diethyl ether:pentane 60:40) 0.75; RI
1
(ZB1/ZB5) 2223/2274; H NMR (400 MHz, CDCl3) d
1.18 (6H, d, J = 7 Hz, CH3·2), 1.78 (3 H, d, J = 1 Hz,
CH3), 1.82 (3 H, s, CH3), 3.36 (2 H, d, J = 7.2 Hz,
@CH–CH2), 3.8 (1H, qq, –CH–(CH3)2), 5.25 (1H, m,
@CH) 5.8 (1 H, s, Carom.H); EIMS, 70 eV, m/z (rel.
int.): 264 (18, [M]+), 221 (39), 209 (6), 191 (3), 177 (3),
165 (100), 153 (5), 69 (5), 55 (3), 43 (3).
4. Experimental
3-(3-Methyl-2-butenyl)-2,4,6-triacetoxyisobutyrophe-
none: RI (ZB1) 2341; EIMS, 70 eV, m/z (rel. int.): 347
(27, [M–C3H7]+), 305 (72, [M–C3H7–1·ketene]+),
277(4) 263 (86, [M–C3H7–2·ketene]+), 245 (13),
221(100, [M–C3H7–3·ketene]+), 207 (6),177 (12), 165
(81), 43 (40).
4.1. Chemicals
Phlorisobutyrophenone was synthesized as described
previously (Klingauf et al., 2005). Phloracetophenone
and 3,3-dimethylallyl bromide were purchased from Ac-
ros Organics (Geel, Belgium) and Fluka (Buchs, Swit-
zerland), respectively. 3,3-Dimethylallyl alcohol and
trichloroacetonitrile were obtained from Aldrich (Stein-
heim, Germany).
4.3. Synthesis of dimethylallylphloracetophenone (3-(3-
methyl-2-butenyl)-2,4,6-trihydroxyacetophenone)
We used the procedure described above, except that
2,4,6-trihydroxyacetophenone (4 mmol) was dissolved
in 5.3 ml aq. KOH (10%) and 10.5 mmol of prenyl bro-
mide were added. The product was purified on a silica
gel column (2.5 · 40 cm) using diethyl ether:pentane
60:40 as solvent. The spectroscopic properties of the
product were consistent with published data (Jakupovic
et al., 1986). The acetylation procedure is described un-
der Section 4.2.
3-(3-Methyl-2-butenyl)-2,4,6-trihydroxyacetophenone:
Rf (silica gel, diethyl ether:pentane 60:40) 0.44; RI (ZB5)
2177; 1H NMR (400 MHz, CDCl3) d 1.78 (3 H, s, CH3),
1.83 (3 H, s, CH3), 2.66 (3 H, s, COCH3), 3.36 (2 H, d,
4.2. Synthesis of dimethylallylphlorisobutyrophenone (3-
(3-methyl-2-butenyl)-2,4,6-trihydroxyisobutyrophenone)
The reaction was conducted according to the method
of Xiao et al. (1998). 2,4,6-Trihydroxyisobutyrophenone
(4.1 mmol) was dissolved in 10 ml aq. KOH (10%) and
cooled to 0 ꢀC. Prenyl bromide (6.1 mmol) was added
dropwise over 5 min. After stirring for 1 h at room tem-
perature, the reaction mixture was poured into 10 ml of
ice water, acidified with HCl (10%) to pH 2 and extracted
with ethyl acetate (3 · 10 ml). The combined organic
phases were dried over Na2SO4 and concentrated under

56
Z. Boubakir et al. / Phytochemistry 66 (2005) 51–57
J = 7.2 Hz, CH–CH2), 5.25 (1H, m, @CH–CH2), 5.83 (1
H, s, Carom.H); EIMS, 70 eV, m/z (rel. int.): 181 (100,
[M–C4H7]+), 236 (88, [M]+), 165 (34), 221 (32, [M–
CH3]+), 193 (32, [M–CH3CO]+), 163 (20), 43 (18), 153
(14), 203 (11), 182 (9), 69 (9),168 (7).
3-(3-Methyl-2-butenyl)-2,4,6-triacetoxyacetophenone:
RI (ZB1) 2248; EIMS, 70 eV, m/z (rel. int.): 319 (40, [M
–CH3CO], 277 (96, [M–CH3CO–1·keten]), 278 (19), 235
(100, [M–CH3CO–2·keten]), 236 (34), 223 (10), 221 (13)
193 (17, [M–CH3CO–3·keten]), 181 (53), 168 (4), 165
(8), 163 (4), 109 (3), 43 (35).
M Tris–HCl, pH 7.5 and 50 ll desalted protein extract
(approx. 100 lg protein). After incubation for 30 min
at 37 ꢀC, the enzymatic products were extracted twice
with ethyl acetate. The combined organic phases were
evaporated to dryness and the residue was dissolved in
50 ll methanol. For large-scale incubations the assay
volume was increased to 4 ml.
Km values were calculated from Lineweaver–Burk
plots. For DMAPP, the concentration was changed
between 0.2 and 16 mM while that of phlorisobutyro-
phenone was kept constant at 2 mM. For phlorisobuty-
rophenone, the concentration varied between 0.1 and 8
mM while the assays contained 2 mM DMAPP. Two
independent experiments were performed and mean val-
ues calculated.
4.4. Synthesis of DMAPP
DMAPP was prepared as described by Cornforth and
Popjak (1969), starting with 3,3-dimethylallyl alcohol (6
mmol), trichloroacetonitrile (36 mmol) and di-triethyl-
ammonium phosphate (14.4 mmol). The reaction prod-
uct was purified on a silica gel 60 (Merck, Darmstadt,
Germany) column (2.5 · 40 cm) using n-propanol:am-
monia:water (6:3:1) as solvent and lyophilized. The
NMR spectra of the product agreed with published data
(Davisson et al., 1986).
4.8. Analytical methods
HPLC of enzymatic products. We used a 1525 Binary
HPLC Pump coupled with a 2487 Dual Absorbance
Detector and equipped with the Breeze software 3.20
(Waters, Eschborn, Germany). The mobile phase con-
sisted of water (A) and methanol (B) on a C18 100-5 col-
Rf (silica gel, n-propanol:ammonia:water 6:3:1) 0.31;
1H NMR (400 MHz, D2O) d 1.71 (3 H, s, CH3), 1.75
(3 H, s, CH3), 4.44 ( 2 H, dd, JH,H = 7 Hz, JH,P = 7
Hz, CH2), 5.44 (1 H, t, JH,H = 7 Hz, (CH3)2–CH); 13C
NMR (100 MHz, D2O) d 19.8, 27.5, 65,2 (d, JC,P = 5.3
Hz), 122.3 (d, JC,P = 8.6 Hz), 142.5; 31P NMR (160
MHz, D2O) d À9.7 (1 P, d, JP,P = 21.6 Hz), À6.9 (1 P,
d, JP,P = 21.6 Hz).
umn (0.4 · 25 cm; Macherey-Nagel, Duren, Germany).
¨
The gradient was as described under Section 4.2. The
flow rate was 1 ml/min and the detection wavelength
290 nm.
Isolation of enzymatic products. Enzyme assays (4 ml)
were extracted with ethyl acetate (3 · 6 ml). After re-
moval of the combined organic phase in vacuo, the res-
idue was dissolved in methanol and separated by TLC
(silica gel 60 F254 (Merck), diethyl ether:pentane
60:40). The zones with Rf 0.75 (dimethylallyl phlori-
sobutyrophenone) and 0.44 (dimethylallyl phloraceto-
phenone) were scraped off. The compounds were
eluted with ethyl acetate, acetylated as described under
Section 4.2 and analyzed by GC–MS.
4.5. Cell cultures
Cell suspension cultures were grown as described pre-
viously (Klingauf et al., 2005).
4.6. Enzyme preparation and ultracentrifugation
GC–MS. Analyses were performed as described pre-
viously (Liu et al., 2004).
The procedure was carried out at 0–4 ꢀC. Three-day-
old cultured cells (5 g) were collected by suction filtra-
tion, mixed with 0.5 g Polyclar AT (Serva, Heidelberg,
Germany) and homogenized in 2.5 ml of 0.1 M sodium
phosphate buffer, pH 6.5 containing 1 mM DTT, using a
Teflon homogenizer. The homogenate was centrifuged
at 10,000g for 15 min and the supernatant either passed
through a PD-10 column (Amersham Biosciences) equil-
ibrated with 0.1 M Tris–HCl pH 7.5 containing 1 mM
DTT or subjected to ultracentrifugation at 100,000g
for 30 min. Protein concentrations were assayed by the
method of Bradford (1976).
Acknowledgements
This research was supported by a scholarship from
the Libyan government (to Z. Boubakir) and a
grant from the Deutsche Forschungsgemeinschaft (to
L. Beerhues).
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