β-peltatin 6-O-methyltransferase from suspension cultures of Linum nodiflorum

Article abstract of DOI:10.1016/S0031-9422(03)00196-1

S-Adenosyl-L-methionine:β-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl-L-methionine to the only free OH-group of β-peltatin in position 6 thus forming β-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 °C. The enzyme activity is strongly inhibited by MnSO ₄, FeCl₃, FeSO₄ and ZnSO₄ as well as S-adenosyl-homocysteine. Mg²⁺ and EDTA did not influence the methylation of β-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and β-peltatin and apparent K_m-values of 15 μM and 40 μM, respectively, were determined for these substrates. Substrate inhibition was observed for β-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:β-peltatin 6-O-methyltransferase. Highest specific activities of β-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.

Full text of DOI:10.1016/S0031-9422(03)00196-1

Phytochemistry 64 (2003) 453–458
www.elsevier.com/locate/phytochem
b-Peltatin 6-O-methyltransferase from suspension cultures
of Linum nodiflorum
Kerstin Kranz, Maike Petersen*
Institut fu¨r Pharmazeutische Biologie, Philipps-Universita¨t Marburg, Deutschhausstr. 17 A, D-35037 Marburg, Germany
Received 15 January 2003; received in revised form 4 March 2003
Dedicated to the memory of Professor Jeffrey B. Harborne
Abstract
S-Adenosyl-l-methionine:b-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum
nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme trans-
fers a methyl group from S-adenosyl-l-methionine to the only free OH-group of b-peltatin in position 6 thus forming b-peltatin-A
methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxy-
ꢀ
podophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 C. The enzyme activity is strongly
inhibited by MnSO , FeCl , FeSO and ZnSO4 as well as S-adenosyl-homocysteine. Mg2+ and EDTA did not influence the
4
3
4
methylation of b-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and b-peltatin and apparent
-values of 15 mM and 40 mM, respectively, were determined for these substrates. Substrate inhibition was observed for b-peltatin.
K
m
No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with
respect to growth, medium alterations and lignan production as well as activity of SAM:b-peltatin 6-O-methyltransferase. Highest
specific activities of b-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the
highest levels of 6-methoxypodophyllotoxin on days 7 to 12.
#
2003 Elsevier Ltd. All rights reserved.
Keywords: Linum nodiflorum; Linaceae; Plant cell cultures; Aryltetralin lignans; b-peltatin; 6-methoxypodophyllotoxin; Lignan biosynthesis;
O-methyltransferase
1
. Introduction
podophyllotoxin 1 itself. Podophyllotoxin 1 and related
compounds are not only present in the family Podo-
phyllaceae, but also in e.g. Juniperaceae, Lamiaceae and
Linaceae (Petersen and Alfermann, 2001). We are cur-
rently concentrating on species of the Linaceae, mainly
the species Linum album, L. flavum and L. nodiflorum L.
(Smollny et al., 1998; Empt et al., 2000; Petersen und
Alfermann, 2001; Kuhlmann et al., 2002). Cell cultures
of these Linum species accumulate considerable
amounts of podophyllotoxin 1 and/or 6-methoxy-
podophyllotoxin 2. Therefore, these cell cultures can
serve as suitable systems for the investigation of the
biosynthesis of the aryltetralin lignans.
It is generally accepted that most lignans are dimers
of hydroxycinnamyl alcohol moieties, e.g. coniferyl alco-
hol. They can be stereospecifically coupled to (+)-pino-
resinol by a system of two proteins called ‘‘pinoresinol
synthase’’ consisting of a laccase and a non-catalytic
‘‘dirigent protein’’ acting together (Davin et al., 1997).
Aryltetralin lignans derived from the naturally occur-
ring podophyllotoxin 1 are important cytotoxic drugs
used in cancer treatment. Currently, podophyllotoxin 1
is isolated from rhizomes of Podophyllum hexandrum
(
Podophyllaceae) growing in the Himalayas, which is
still collected from the wild and therefore counts among
the endangered species. Podophyllotoxin 1 is then semi-
synthetically transformed to e.g. Teniposide, Etoposide
1
and Etopophos . These compounds act cytotoxically
by binding to DNA/topoisomerase II complexes and
thus inducing DNA strand breakages (Damayanthi and
Lown, 1998; Imbert, 1999). This is in contrast to the
microtubule-inhibiting activity of the parent compound
*
Corresponding author. Tel.: +49-6421-2825821; fax: +49-6421-
825828.
E-mail address: petersen@mailer.uni-marburg.de (M. Petersen).
2
0
031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00196-1

454
K. Kranz, M. Petersen / Phytochemistry 64 (2003) 453–458
(
secoisolariciresinol by pinoresinol/lariciresinol reductase
+)-Pinoresinol is reduced via (+)-lariciresinol to (ꢁ)-
methylated in position 6 to b-peltatin-A methylether 5
and finally hydroxylated in position 7 to 6-methoxy-
podophyllotoxin 2. 6-Methoxypodophyllotoxin 2 as well
as podophyllotoxin 1 in cells accumulating this lignan
are safely stored in the vacuole as glucosides (Henges et
al., 1999) thus preventing cytotoxic effects on the produ-
cing cells. We here describe the identification and charac-
terization of a new enzyme in plant secondary metabolism,
S-adenosyl-l-methionine:b-peltatin 6-O-methyltransferase
(b-peltatin 6OMT), from Linum nodiflorum suspension
cultures, the S-adenosylmethionine-dependent methyl-
transferase responsible for the transformation of b-pel-
tatin 4 to b-peltatin-A methylether 5.
(Dinkova-Kostova et al., 1996) and then oxidized to
(ꢁ)-matairesinol by secoisolariciresinol dehydrogenase
(Xia et al., 2001). The biosynthetic pathway from
matairesinol to the aryltetralin lignans is not yet clari-
fied. Feeding experiments with plant cell cultures sug-
gested that deoxypodophyllotoxin
3 is the first
aryltetralin lignan and the common precursor for the
biosynthesis of both, podophyllotoxin 1 and 6-methoxy-
podophyllotoxin 2 (Van Uden et al., 1995, 1997). These
two lignans 1, 2 are the main lignans in the above-men-
tioned Linum species where they are stored as glucosides
in the vacuole (Henges, 1999). Other experiments with
Linum flavum and Podophyllum peltatum suggested a
biosynthetic pathway via 7-hydroxymatairesinol to
2. Results and discussion
6
-methoxypodophyllotoxin 2 (Xia et al., 2000). First
hints towards the enzyme hydroxylating deoxy-
podophyllotoxin 3 in position 7 to podophyllotoxin 1
were found by Henges (1999) in microsomal prepara-
tions from cell cultures of Linum album. The enzyme
introducing a hydroxyl group in position 6 of deoxy-
podophyllotoxin 3 forming b-peltatin 4 was detected
and characterized from cell cultures of Linum flavum
and L. nodiflorum. This deoxypodophyllotoxin
2.1. Identification and characterization of
S-adenosylmethionine:ꢀ-peltatin 6-O-methyltransferase
According to precursor administration, the biosyn-
thetic pathway from deoxypodophyllotoxin
3 to
6-methoxypodophyllotoxin 2 follows the steps (1)
hydroxylation of deoxypodophyllotoxin 3 to b-peltatin
4, (2) methylation of b-peltatin 4 to b-peltatin-A
methylether 5 and (3) 7-hydroxylation of the latter to
6-methoxypodophyllotoxin 2 (Van Uden et al., 1995,
1997) (Fig. 1). Previous studies have shown the presence
6-hydroxylase (DOP6H) was characterized as a cyto-
chrome P450 enzyme system (Molog et al., 2001; Kuhl-
mann et al., 2002). The product b-peltatin 4 is further
Fig. 1. Hypothetical scheme for the biosynthesis of the aryltetralin lignans podophyllotoxin 1 and 6-methoxypodophyllotoxin 2 from deoxy-
podophyllotoxin 3; (1)=deoxypodophyllotoxin 7-hydroxylase, (2)=deoxypodophyllotoxin 6-hydroxylase, (3)=SAM:b-peltatin 6-O-methyl-
transferase, (4)=b-peltatin-A methylether 7-hydroxylase.

K. Kranz, M. Petersen / Phytochemistry 64 (2003) 453–458
455
of deoxypodophyllotoxin 6-hydroxylase in cell cultures
of Linum flavum and L. nodiflorum that catalyzes the
formation of b-peltatin 4, the 6-hydroxy substrate for
this hence uncharacterized O-methyltransferase reaction
mM showing a high affinity for this substrate. The Km-
values reported for SAM for different plant O-methyl-
transferases vary over a wide range. Apparent Km-
values as low as 3 mM were determined for caffeate
O-methyltransferase from Nicotiana tabacum (Collen-
davelloo et al., 1981) and Medicago sativa root nodules
(Vance and Bryan, 1981) or 10-hydroxydihydro-
sanguinarine 10-O-methyltransferase from Eschscholtzia
californica (De Eknamkul et al., 1992), values as high as
(Molog et al., 2001; Kuhlmann et al., 2002). O-Methyl-
ation reactions in plant secondary metabolism are
mostly dependent on S-adenosylmethionine (SAM) as
methyl donor and therefore we incubated protein pre-
parations from cell cultures of Linum nodiflorum with
SAM and b-peltatin 4 and were able to demonstrate the
formation of b-peltatin-A methylether 5 by HPLC ana-
lysis. The reaction product was identified by co-chroma-
tography with authentic b-peltatin-A methylether 5 on
different columns (Hypersil ODS, Hypersil HyPurity
Elite C ) and with different solvent systems (30–50%
0
160 mM were reported for isoflavone 4 -O-methyl-
transferase from Cicer arietinum (Wengenmayer et al.,
1974). S-Adenosylhomocysteine (SAH) strongly inhib-
ited the methylation reaction in a concentration-depen-
dent manner. Inhibition curves indicate competitive
inhibition by SAH (which is a reaction product) as was
already observed for other O-methyltransferases, for
example caffeate O-methyltransferase from Beta vulgaris
1
8
acetonitrile; 60% methanol). Furthermore, diode array-
detection showed identical retention times and
UV-spectra of the reaction product and authentic
b-peltatin-A methylether 5. The same protein prepara-
tions did not show methylation of caffeic acid to ferulic
acid excluding side activity of caffeic acid O-methyl-
transferase. The enzyme is soluble and can be pre-
cipitated between 40 and 80% saturation of ammonium
sulfate; this, however, often results in severe loss of
enzyme activity. The enzyme activity is essentially
dependent on the addition of the substrates b-peltatin 4
and SAM, but no other co-factors are needed such as
for example Mg2+ which has no effect on the activity of
0
(Poulton and Butt, 1975) or isoflavone 4 -O-methyl-
transferase from Cicer arietinum (Wengenmayer et al.,
1974).
The substrate specificity was determined using differ-
ent potential substrates from the lignan biosynthetic
pathway carrying a free OH-group (e.g. podophyllo-
toxin 1, matairesinol, pinoresinol) as well as caffeic acid.
A methylation reaction was only observed for b-peltatin
4. For this substrate Michaelis–Menten-kinetics were
determined with a marked substrate inhibition at con-
centrations higher than 100 mM b-peltatin 4 (Fig. 2).
2
+
b-peltatin 6OMT. Mg markedly stimulated the activity
of e.g. caffeoyl-CoA O-methyltransferase from Daucus
The apparent K -value for b-peltatin 4 was at 40 mM
m
(Fig. 2 insert).
¨
carota (Kuhnl et al., 1989) or Petroselinum crispum
Pakusch et al., 1989) and is required for different
quercetin O-methyltransferases from Chrysosplenium
americanum (Ibrahim and De Luca, 1982). Corres-
pondingly, EDTA has no influence on the b-peltatin
(
2.2. Characterization of a suspension cell line of Linum
nodiflorum
A callus culture, and subsequently a suspension cul-
ture, was established from aseptically germinated seed-
lings of Linum nodiflorum (Konuklugil et al., 1999). The
cell line used in this study consisted of relatively large
brownish aggregates. This cell line was characterized for
6
OMT activity. Other ions showed inhibiting activities:
1
down to a residual activity of 35%. Concentrations
0 mM MnSO inhibited the methylation of b-peltatin 4
4
higher than 5 mM FeSO or FeCl resulted in activities
4
3
lower than 10% of the control. Strongest inhibition was
observed for ZnSO which completely inhibited b-pel-
4
tatin 6OMT activity at 5 mM. Similar effects have been
observed for other O-methyltransferases from plants,
0
e.g. isoflavone 4 -O-methyltransferase from Cicer arieti-
num (Wengenmayer et al., 1974) or norcoclaurine 6-O-
methyltransferase from Coptis japonica (Sato et al.,
1
reaction was determined to be at 40 C with a steep
994). The temperature optimum of the methylation
ꢀ
ꢀ
decline of the activity above 45 C. For this reason
ꢀ
standard assays were performed at 38 C. The pH-opti-
mum in Tris/HCl-buffer is at pH 7.7 with half-maximal
activities at pH 6.2and pH 8.5.
b-Peltatin 6OMT uses SAM as methyl donor. The
enzyme showed Michaelis–Menten-kinetics for this
substrate and was saturated at a concentration of 150
Fig. 2. Substrate saturation curve for b-peltatin 4 showing the sub-
strate inhibition at concentrations higher than 100 mM; insert: Line-
weaver–Burk-diagram for the determination of the apparent K
at 40 mM.
m
-value
mM SAM. The apparent K -value was determined at 15
m

456
K. Kranz, M. Petersen / Phytochemistry 64 (2003) 453–458
1
5 days with respect to growth, alteration of the med-
7 and then decreases slowly. As alterations in the culture
medium, the sugar content, the pH and the conductivity
were measured (Fig. 3B). Sugar consumption is seen by
the decrease of the refractive index; the sugar content is
very low from day 7 onwards and reaches a minimum
value at day 9. Similarly ion concentrations as deter-
mined by conductivity measurements decreased until
day 9. The changes observed after day 9 are due to cell
lysis and cell death. The declining growth and death of
the Linum cells is clearly correlated to the depletion of
sugar from the medium since the stationary phase of
fresh weight accumulation starts at day 7. The pH of the
culture medium increases nearly constantly from pH 4.8
at the beginning of the culture period to about pH 7.6.
Lignans were isolated from the cells and quantified as
the respective aglyca (Fig. 3C). Podophyllotoxin 1 and
b-peltatin-A methylether 5 were not detected in these
cell extracts. Deoxypodophyllotoxin 3, the putative
common precursor of podophyllotoxin 1 and 6-methoxy-
podophyllotoxin 2, is present in the cells in quite high
levels (0.08 to 0.18% of the cell dry weight) throughout
the culture period. b-Peltatin 4 is only found in the sec-
ond half of the culture period at higher levels. An
increase is seen from day 6 onwards. Low levels of
ium composition, lignan accumulation and activity of
b-peltatin 6OMT. Growth was measured as fresh (FW)
and dry weight (DW) accumulation (Fig. 3A). Fresh
weight increased with a slight lag phase from the begin-
ning of the culture period until day 10 and declined
afterwards. Dry weight increases linearly from day 0 to
6-methoxypodophyllotoxin 2 can be determined until
day 6 of the culture period. Then the levels increase and
show values around 0.15% of the cell dry weight from
days 7 to 12after which a strong decrease is observed
which might be caused by degradation but is most
probably due to cell lysis. Aryltetralin lignans could be
extracted and determined from used culture medium
(data not shown). In a different cell line of Linum nodi-
florum, up to 1.7% 6-methoxypodophyllotoxin 2 in the
cell dry weight were measured on day 9 of the culture
period (Kuhlmann et al., 2002). The specific activity of
b-peltatin 6OMT (Fig. 3A) shows a short increase just
after inoculation of the cell culture and then the activity
decreases until day 3. A strong increase in specific
activity is observed until day 7, and then decreases until
the end of the culture period. This correlates with the
accumulation of 6-methoxypodophyllotoxin 2. Attempts
to enhance the activity of b-peltatin 6OMT by addition
of 50 and 100 mM methyl jasmonate to the cultures were
not successful. This correlates with earlier findings that
methyl jasmonate as well as other well-known elicitors
did not enhance the accumulation of aryltetralin lignans
in Linum cell cultures (data not shown).
3. Conclusion
Fig. 3. Characterization of a suspension culture of Linum nodiflorum.
A: Growth measured as fresh (FW, *) and dry weight (DW, *) accu-
mulation and specific activity of b-peltatin 6OMT (&); B: Sugar content
Suspension cultures of Linum nodiflorum L. (Linaceae)
accumulate 6-methoxypodophyllotoxin(-glucoside) as
one of the main cytotoxic lignans. The hypothetical
biosynthetic pathway goes from deoxypodophyllotoxin
3 which is hydroxylated in position 6 by a cytochrome
(&), pH (&) and conductivity (*) in the culture medium; C: Lignan
content in the cells determined as aglyca: deoxypodophyllotoxin 3 (*),
b-peltatin 4 (&) and 6-methoxypodophyllotoxin 2 (&); podophyllotoxin
1
and b-peltatin-A methylether 5 were not found in the cell extracts.

K. Kranz, M. Petersen / Phytochemistry 64 (2003) 453–458
457
P450 monooxygenase (deoxypodophyllotoxin 6-hyd-
4.3. Assay for ꢀ-peltatin 6-O-methyltransferase
roxylase) to b-peltatin 4. b-Peltatin 4 is then methylated
to b-peltatin-A methylether 5 by SAM:b-peltatin 6-O-
methyltransferase, now detected and characterized from
cell cultures of Linum nodiflorum. The highest activity of
this enzyme was found on day 7 of the culture period
and is followed by the accumulation of 6-methoxy-
podophyllotoxin(-glucoside) at days 7 to 12in the
Linum cells. These findings support the suggested hypo-
thetical biosynthetic pathway from deoxypodophyllo-
toxin 3 via b-peltatin 4 and b-peltatin-A methylether 5
to 6-methoxypodophyllotoxin 2.
The assays contained 150 mM b-peltatin 4, 2 00mM
S-adenosyl-l-methionine and approximately 150 mg
protein in a total volume of 250 ml 0.1 mM Tris/HCl
ꢀ
(pH 7.5). They were incubated at 38 C for 2to 3 h (1
h for the determination of K -values). The reaction
m
was stopped by adding 6 N HCl (50 ml). The assays
were extracted with EtOAc (3ꢂ500 ml). The solvent
was evaporated under vacuum and the residue resus-
pended in 50% aq. MeOH (50 ml) and analyzed by
HPLC. The HPLC system consisted of a HyPurity
Elite C18 column, 50% aq. MeCN as mobile phase at a
flow of 1.25 ml/min. The detection wavelength was 280
nm. 0.1 mM b-peltatin-A methylether 5 was used as
standard.
4
. Experimental
4
.1. Plant material
4.4. Inhibition of ꢀ-peltatin 6-O-methyltransferase
Callus cultures of Linum nodiflorum were established
r Pflanzengenetik
from seeds provided by the Institut fu
¨
For the tests for inhibitors the standard assay was
modified by adding 0 to 10 mM of MgSO , MgCl ,
4 2
und Kulturpflanzenforschung (Gatersleben, Germany).
Cell suspension cultures of Linum nodiflorum were
derived from these callus cultures by transferring 5 g
callus cells to 50 ml medium in a 250 ml Erlenmeyer
flask (Konuklugil et al., 1999). Standard medium for
callus and suspension cultures was MS-medium (Mura-
MnSO , ZnSO , FeSO and FeCl . S-Adenosyl-homo-
4
4
4
3
cysteine as an inhibitor was added to the assays in a
concentration range from 0 to 400 mM.
4.5. Characterization of suspension cultures of Linum
nodiflorum
ꢁ
1
shige and Skoog, 1962) containing 30 g l sucrose and
ꢁ
1
0
.4 mg l naphthalene acetic acid. Suspension cultures
are grown in continuous light with shaking at 125 rpm.
The suspension cultures are subcultivated every 7 days
by transferring 5 g wet cells with a perforated spoon
into 50 ml fresh medium.
Suspension cultures of L. nodiflorum were inoculated
as described above and cultivated under standard con-
ditions for 16 days. Every day one to three flasks were
harvested by suction filtration and the fresh weight
ꢀ
recorded. Fresh cells (5 g) were frozen at ꢁ20 C and
4
.2. Preparation of protein extracts
lyophilized for 48 h for the determination of the dry
weight and for lignan extraction. The remaining fresh
cells were frozen in liquid nitrogen and stored at
Suspension cells (7-day old) of Linum nodiflorum were
ꢀ
separated from the medium by suction filtration. After
addition of 1 ml buffer (0.1 M Tris/HCl, 1 mM dithio-
threitol, pH 7.5; for the determination of the pH-opti-
mum: 0.01 M Tris/HCl, 1 mM dithiothreitol, pH 7.5)
and 0.1 g Polyclar 10 per 1 g cell fresh weight, the cells
were homogenized (3ꢂ30 s) using an Ultraturrax (Janke
and Kunkel, Freiburg im Breisgau, Germany) with
ꢁ20 C before extraction for enzyme assays as descri-
bed above. In the cell-free medium, the pH and con-
ductivity were measured with appropriate electrodes.
The sugar content was determined by measuring the
refractive index.
Lignans were extracted from powdered lyophilized
cells (200 mg) with MeOH (2 ml). The mixture was
further homogenized in an ultrasonic bath (2ꢂ30 s)
with intermediate cooling on ice and then diluted with
H O (8 ml; adjusted to pH 5 with H PO ). After
ꢀ
intermediate cooling on ice. After centrifugation (4 C,
1
2000 g, 20 min) the supernatant was used as crude
enzyme preparation. For most assays (except culture
characterization) the protein fraction precipitating
between 40 and 80% saturation of ammonium sulfate
was used. Precipitated protein was sedimented (48,000
2
3
4
addition of
1
mg b-glucosidase (from almonds,
>1000 U mg ; Roth, Germany) the mixture was
ꢁ
1
ꢀ
incubated at 35 C for 3 h. Then CH Cl (10 ml)
2
2
ꢀ
g, 4 C, 20 min), redissolved in 0.1 M Tris/HCl, 1 mM
was added and mixed vigorously for 20 min. After
centrifugation the CH Cl phase was collected and
dithiothreitol, pH 7.5 and desalted by gel filtration on
PD-10 columns (Amersham Biosciences, Freiburg im
Breisgau, Germany) using the same buffer. Protein
concentrations were determined according to Bradford
2
2
evaporated. The residue was dissolved in MeOH
(3ꢂ500 ml) and lignan composition was analyzed by
HPLC as described above with 40% and 50% aq.
MeCN as mobile phase using appropriate pure lignans
as standards.
ꢁ
(
standard.
1976) using bovine serum albumin (1 mg ml 1) as

4
58
K. Kranz, M. Petersen / Phytochemistry 64 (2003) 453–458
M., 2002. Aspects of cytotoxic lignan biosynthesis in suspension
Acknowledgements
cultures of Linum nodiflorum. Phytochemistry Reviews 1, 37–43.
Molog, G.A., Empt, U., Kuhlmann, S., Van Uden, W., Pras, N.,
Alfermann, A.W., Petersen, M., 2001. Deoxypodophyllotoxin
b-peltatin 4 and b-peltatin-A methylether 5 were a
kind gift from Dr. Manuel Medarde, University of
Salamanca (Spain). Cell cultures of Linum nodiflorum
were kindly donated by Prof. Dr. A.W. Alfermann,
6
-hydroxylase, a cytochrome P450 monooxygenase from cell cultures
of Linum flavum involved in the biosynthesis of cytotoxic lignans.
Planta 214, 288–294.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth
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¨
University of Dusseldorf (Germany).
4
97.
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methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase from
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