Báo cáo khóa học: Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-methyltransferase ppt

Furanocoumarin biosynthesis in
Ammi majus
L.
Cloning of bergaptol
O
-methyltransferase
Marc Hehmann
1,
*, Richard Lukac
ˇ
in
1,
*, Halina Ekiert
2
and Ulrich Matern
1
1
Institut fu
¨
r Pharmazeutische Biologie, Philipps-Universita
¨
t Marburg, Germany;
2
Department of Pharmaceutical Botany,
Collegium Medicum, Jagiellonian University, Krako
´
w, Poland
Plants belonging to the Apiaceae or Rutaceae accumulate
methoxylated psoralens, such as bergapten or xanthotoxin,
as the final products of their furanocoumarin biosynthesis,
and the rate of accumulation depends on environmental and

other cues. Distinct O-methyltransferase activities had been
reported to methylate bergaptol to bergapten and xantho-
toxol to xanthotoxin, from induced cell cultures of Ruta
graveolens, Petroselinum crispum and Ammi majus. Bergap-
tol 5-O-methyltransferase (BMT) cDNA was cloned from
dark-grown Ammi majus L. cells treated with a crude fungal
elicitor. The translated polypeptide of 38.7 kDa, composed
of 354 amino acids, revealed considerable sequence similar-
ity to heterologous caffeic acid 3-O-methyltransferases
(COMTs). For homologous comparison, COMT was
cloned from A. majus plants and shown to share 64%
identity and about 79% similarity with the BMT sequence at
the polypeptide level. Functional expression of both enzymes
in Escherichia coli revealed that the BMT activity in the
bacterial extracts was labile and rapidly lost on purification,
whereas the COMT activity remained stable. Furthermore,
the recombinant AmBMT, which was most active in
potassium phosphate buffer of pH 8 at 42 °C, showed
narrow substrate specificity for bergaptol (K
mSAM
6.5 l
M
;
K
m Bergaptol
2.8 l
M
) when assayed with a variety of sub-
strates, including xanthotoxol, while the AmCOMT accep-
ted 5-hydroxyferulic acid, esculetin and other substrates.

Dark-grown A. majus cells expressed significant BMT
activity which nevertheless increased sevenfold within 8 h
upon the addition of elicitor and reached a transient maxi-
mum at 8–11 h, whereas the COMT activity was rather low
and did not respond to the elicitation. Complementary
Northern blotting revealed that the BMT transcript abun-
dance increased to a maximum at 7 h, while only a weak
constitutive signal was observed for the COMT transcript.
The AmBMT sequence thus represents a novel database
accession specific for the biosynthesis of psoralens.
Keywords: Ammi majus L.; Apiaceae; furanocoumarin
biosynthesis; bergaptol O-methyltransferase; caffeate
O-methyltransferase.
Cell suspension cultures of the Apiaceae, in particular Ammi
majus L. [1,2] and Petroselinum crispum [3–6], have served
in numerous model studies on the induced plant disease
resistance response. Upon treatment with fungal elicitor,
these cells produce linear furanocoumarins (psoralens)
besides lignin-like compounds for reinforcement of their cell
walls [7–9]. Various crude cell wall elicitors, particularly Pmg
(from Phythophthora sojae,formerlyPhythophthora mega-
sperma f. sp. glycinea), have been used in the past, and at least
in case of the induction of parsley cells the eliciting principle
has been identified as a peptide [10]. Furanocoumarins are
potentially toxic compounds which probably function as
phytoalexins in the response to fungal infection [3], but their
accumulation can also be triggered by other means, e.g.
wounding of plants or exposure to acid fog [11,12]. A
considerable proportion of psoralens may be recovered from
the surface of plants [13], and most of the psoralens elicited in

cell cultures also accumulated in the culture fluid. Psoralens
are capable of intercalating with DNA, and the methoxyl-
ated psoralens bergapten and xanthotoxin are the most
relevant natural furanocoumarins in terms of their thera-
peutic potential. These psoralens exhibit photosensitizing
and antiproliferative activities [14] and were evaluated as
photosensitizing drugs in oral psoralen plus UVA irradiation
(PUVA) therapy of psoriasis and vitiligo [15,16].
A. majus cells also uniquely produce derivatives of 7-O-
prenylumbelliferone under conditions of elicitation (Fig. 1)
[17], and the induction of coumarin biosynthesis in these cell
cultures provided the basis for extensive in vitro investiga-
tions. Both the umbelliferone 7-O-prenyltransferase activity
and a 6-C-prenyltransferase activity forming demethylsube-
rosin en route to the psoralens (Fig. 1) were found
associated with the microsomal fraction [18]. Such a 6-C-
prenyltransferase activity had been reported initially as a
Mn
2+
-dependent enzyme from Ruta graveolens and
assigned to the plasitidic membranes [19]. Individual
Correspondence to U. Matern, Institut fu
¨
r Pharmazeutische Biologie,
Philipps-Universita
¨
t Marburg, Deutschhausstrasse 17A,
D-35037 Marburg, Germany.
Fax: + 49 6421 282 6678, Tel.: + 49 6421 282 2461,
E-mail: matern@staff.uni-marburg.de

Abbreviations:SAM,S-adenosyl-
L
-methionine; BMT, bergaptol
5-O-methyltransferase; XMT, xanthotoxol 8-O-methyltransferase;
OMT, O-methyltransferase; COMT, caffeic acid 3-O-methyltrans-
ferase; PUVA, psoralen plus UVA irradiation; RACE, rapid
amplification of cDNA ends; RLM-RACE, RNA ligase-mediated
rapid amplification of cDNA ends.
*These authors contributed equally to the work described.
(Received 3 November 2003, revised 17 December 2003,
accepted 15 January 2004)
Eur. J. Biochem. 271, 932–940 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03995.x
cytochrome P450 monooxygenases sequentially convert
demethylsuberosin to (+)-marmesin and psoralen, and
these activities were also demonstrated in the microsomal
fraction of elicited A. majus cells [1,2]. Moreover, the
conversion of (+)-marmesin to psoralen (Fig. 1) was
proven to proceed by syn-elimination releasing acetone
[20]. The subsequent hydroxylation of psoralen in the 5- or
8-position yields bergaptol and xanthotoxol, respectively
(Fig. 1). Both of these hydroxylations to yield 5,8-dihyd-
roxypsoralen are required for the formation of isopimpin-
ellin, which commonly accumulates as a minor byproduct
upon induction, but the order of hydroxylations and
O-methylations remains unresolved [19]. However, psoralen
5-monooxygenase activity forming bergaptol was demon-
strated in vitro with microsomes from elicited A. majus cells
[2]. The biosynthesis of bergaptol from umbelliferone is
thus entirely catalyzed through membrane-bound enzymes,
involving one prenyltransferase and three P450 monoxy-

genases, and is preceded by the formation of umbelliferone
from 4-coumaric acid, which was also proposed to depend
on a P450 monooxygenase. The 5- or 8-hydroxylated
furanocoumarins (bergaptol or xanthotoxol) are further
processed by O-methylation to bergapten and xanthotoxin,
and the corresponding O-methyltransferases (OMTs) were
identified as distinct entities and purified by affinity
chromatography from Ruta graveolens [21] and later also
from Petroselinum crispum [22]. S-Adenosyl-
L
-methionine–
bergaptol 5-O-methyltransferase (SAM–BMT) and S-aden-
osyl-
L
-methionine–xanthotoxol 8-O-methyltransferase
(SAM–XMT) (Fig. 1) are also expressed in dark-grown
A. majus cells, and in all instances these methyltransferases
are soluble and inducible enzymes.
We report here the cloning and functional characteriza-
tion of BMT from elicitor-treated Ammi majus L. cells as a
major step towards a molecular understanding of psoralen
biosynthesis. For comparison, the closely related S-adeno-
syl-
L
-methionine–caffeate 3-O-methyltransferase (SAM–
COMT) was also cloned from A. majus plants, and the
differential regulation of these methyltransferases was
examined upon elicitation of the cell cultures.
Materials and methods
Ammi majus

cell cultures and induction
Cell suspension cultures of A. majus L. (40 mL B5
+
-
medium in 250 mL flasks) were initiated and grown
continuously in the dark as described elsewhere [1,2]. Pmg
Fig. 1. Schematic outline of linear furanocou-
marin biosynthesis. The sequence of hydroxy-
lations and O-methylations of psoralen
leading to isopimpinellin has not been
established.
Ó FEBS 2004 Bergaptol O-methyltransferase in Ammi majus
1
(Eur. J. Biochem. 271) 933
elicitor was suspended in distilled water (5 mgÆmL
)1
), the
suspension was heated to boiling point and added to 6-day-
old cell cultures (1 mL per 40 mL culture). The cells were
harvested 4 h later and immediately frozen in liquid
nitrogen and stored at )70 °C until use.
Chemicals
Biochemicals were purchased from Roth (Karlsruhe,
Germany), vectors and Escherichia coli host strains from
Invitrogen (Karlsruhe, Germany) or Qiagen (Hilden,
Germany). Restriction enzymes and DNA modifying
enzymes were from MBI-Fermentas (St. Leon-Rot,
Germany), Promega (Mannheim, Germany) or Stratagene
(Heidelberg, Germany). Bergaptol was bought from
Extrasynthese (Genay, France), caffeic acid from Roth

(Karlsruhe, Germany), and [methyl-
14
C]S-adenosyl-
L
-methionine was purchased from Hartmann Analytic
(Braunschweig, Germany).
RNA isolation, PCR cloning and heterologous
expression
Total RNA was isolated from Pmg elicitor-induced cells
following the protocol of Giuliano et al. [23]. The time
of elicitor-induction was chosen from previous induction
experiments in which the time course of furanocoumarin-
specific enzyme activities in A. majus had been monitored
[1,2]. Alternatively, the RNA was isolated from the stems
and leaves of 4–6 week-old A. majus plants. cDNA frag-
ments were generated by RT-PCR amplification using
degenerate oligonucleotide primers [24] which had been
designed according to conserved amino acid sequences of
plant OMTs [25,26]. The cDNA fragments were cloned,
sequenced, and full length clones were generated by the
rapid ampification of cDNA ends (RACE) and RNA
ligase-mediated rapid amplification of cDNA ends (RLM-
RACE) techniques, respectively, using gene-specific pri-
mers. Cloning of the PCR products was performed by
TOPO TA Cloning (Invitrogen, Karlsruhe, Germany).
Briefly, the protein coding regions of the putative BMT
and COMT were amplified with 5¢-primers providing an
NcoI site directly before the start codon and 3¢-primers
inserting a BamHI site after the stop codon before they were
cloned into the pCR2.1-TOPO vector. An internal NcoIsite

contained in the ORF of the COMT was deleted by using
QuikChangeÒ Multi Site-Directed Mutagenesis Kit as
described by the manufacturer (Stratagene, Heidelberg,
Germany) without altering the amino acid sequence. The
mutation was verified by DNA sequencing [27], and the
BMT- and COMT-coding DNA clones were subsequently
isolated by digestion with NcoIandBamHI. The cDNAs
were subcloned into pQE60 vector (Qiagen, Hilden,
Germany) for functional expression in E. coli strain M15
(Qiagen) harboring the plasmid pRep4 and employed for
BMT and COMT activity assays, respectively. The expres-
sion was induced by the addition of 1.0 m
M
isopropyl thio-
b-
D
-galactoside [28]. Following the induction, the cells were
harvested by centrifugation [29], disrupted by ultrasonica-
tion, the crude extract was cleared by centrifugation
(30 000 g, 4 °C, 10 min), and enzyme assays were carried
out with the supernatants.
Sequence analysis
The cDNAs amplified by RT-PCR were sequenced by the
dideoxy nucleotide chain termination technique [27]. The
cDNA sequences were subjected to BLAST searches
(advanced WU-Blast2; EMBL) and alignments with
CLUSTALW
algorithm (EMBL).
Purification procedure
The crude extract was fractionated by ammonium sulfate

precipitation from 0 to 45%, 45–60% and 60–80%
saturation. The 60–80% fraction (BMT) and the
45–60% fraction (COMT) were dissolved in 200 m
M
potassium phosphate buffer of pH 8.0 and 200 m
M
Tris/
HCl buffer of pH 7.5, respectively. The extracts were
desalted by size exclusion chromatography
2
through PD-10
columns (Amersham, Freiburg, Germany) and on Frac-
togel EMD BioSEC (S) (Merck, Darmstadt, Germany).
The purification was monitored by SDS/PAGE [30] and
enzyme activity assays of individual fractions.
Enzyme assays and other analytical methods
BMT activity was routinely measured at 42 °C in 200 m
M
potassium phosphate buffer pH 8.0 in the presence of
sodium ascorbate (20 m
M
), magnesium chloride (1.5 m
M
),
bergaptol (250 l
M
)andtherecombinantbacterial
enzyme extract. The reaction was started by the addition
of S-adenosyl-
L

-[methyl-
14
C]methionine (40 l
M
), and the
product was identified as bergapten by silica thin-layer
chromatography employing trichloromethane/ethylacetate
(2 : 1, v/v; R
F bergaptol
0.52, R
F bergapten
0.77), trichlorometh-
ane/methanol (95 : 5, v/v; R
Fbergaptol
0.26, R
F bergapten
0.78),
n-hexane/ethylacetate/methanol (5 : 5 : 1, v/v/v; R
Fbergaptol
0.67, R
F bergapten
0.78) or toluene/ethylacetate (3 : 2, v/v;
R
Fbergaptol
0.34, R
F bergapten
0.68)asthesolventsystems.The
COMT activity assay was carried out at 32 °C in 200 m
M
potassium phosphate buffer pH 7.5 containing sodium

ascorbate (20 m
M
), magnesium chloride (1.5 m
M
), caffeic
acid (250 l
M
)andS-adenosyl-
L
-[methyl-
14
C]methionine
(40 l
M
) in addition to
3
the crude enzyme protein. The
reactions were stopped by the addition of 1
M
4
HCL (30 lL)
and extracted with 400 lL ethyl acetate. Aliquots of the
organic phase (200 lL) were mixed with 5 mL scintillation
cocktail (Roth, Karlsruhe, Germany) and measured in a
liquid scintillation counter (1214 Rackbeta; PerkinElmer,
Wellesley, MA, USA). Incubations with boiled enzyme
(5 min at 100 °C), or mixtures lacking bergaptol and caffeic
acid, were run for control and served for background
corrections.
Linear conditions for kinetic assays were established by

adjusting the amount of enzyme protein (BMT: 0.15–3.0 lg
per assay; COMT: 0.5–10.25 lg per assay). The BMT assays
were usually conducted for 20 min, using 1.5 lgdesalted
protein and 4.0 nmol S-adenosyl-
L
-[methyl-
14
C]methionine
and 25.0 nmol bergaptol per 100 lL incubation, which
secured linear conversion rates for about 60 min. The
COMT assays were carried out accordingly using 5 lg
desalted protein. Protein was determined according
Lowry et al. [31] and the data were extrapolated from
Lineweaver–Burk plots.
934 M. Hehmann et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Northern blotting
Following the addition of Pmg elicitor to the A. majus cell
suspensions, total RNA was isolated from the cells every
0.5 h up to 8 h and used for Northern blot analysis (RNA
dot blot). The RNA (4 lg) was denaturated in 0.5· Mops
buffer pH 6.0, containing 50% (v/v) formamide and 2.2
M
formaldehyde, and transferred to a Hybond-N
+
nylon
membrane (Amersham Biosciences, Freiburg) [32] in an
I-SRc 96-Dot Blot Minifold (Schleicher and Schu
¨
ll, Dassel,
Germany). The full size A. majus BMT cDNA (1062 bp)

and COMT cDNA (1095 bp) were
32
P-labeled using a
Rediprime
TM
II-random prime labeling system (Amersham
Biosciences, Freiburg), and used as probes. The blots were
blocked and then hybridized with 25 ng of one of these two
labeled probes. Hybridization was carried out overnight
at 68 °Cin2· Denhardt’s solution in the presence of 0.5%
(w/v) SDS and 100 lgÆmL
)1
salmon sperm DNA (Sigma,
Deisenhofen, Germany). After stringent washings for
20 min at room temperature in 2· NaCl/Cit followed by
15 min at 68 °Cin2· NaCl/Cit the membranes were
exposed to a Bio Imaging Analyzer FLA-2000 (Fujifilm).
Results
Induction of
O
-methyltransferase activities
Elicitor–induction studies had been carried out previously
with A. majus cell cultures, and the coumarin-specific
enzyme activities (dimethylallyldiphosphate: umbelliferone
6-C-and7-O-dimethylallyltransferases) commonly reached
a first maximum at 12 h of induction [18]. This time course
suggested maximal transcript abundances within the first
6 h of elicitation and corresponded to the patterns reported
for the elicitor induction of phenylalanine ammonia lyase
and 4-coumarate:CoA ligase activities in Petroselinum

crispum cell cultures [33]. However, a different induction
profile was reported by Hauffe et al. [22] for the BMT
activity in P. crispum cells with a maximum beyond 25 h.
Therefore, preliminary assays of BMT and XMT activities
were conducted with crude extracts of dark-grown A. majus
cells and under the conditions described for Ruta graveolens
[34]. These assays revealed that both O-methyltransferase
activities were expressed already in the controls (0.4 lkatÆ
kg
)1
BMT and 66 nkatÆkg
)1
XMT, on average), but
increased considerably in response to treatment of the cell
suspensions with Pmg elicitor. The activities were measured
every three hours over the time period from 2 to 23 h
following the addition of elicitor, and shown to increase
sevenfold within 8 h to reach a transient maximum at about
8–11 h. (H. Ekiert and R. Lukac
ˇ
in, unpublished data).
Thus, the cells at 4 h after the addition of elicitor were
considered to contain the highest BMT and XMT transcript
abundances.
The activity of COMT in A. majus was examined for
comparison, and only low levels (2.5 lkatÆkg
)1
on average)
were observed in crude extracts of suspension-cultured cells,
which hardly changed upon elicitation and might be due to

related OMTs with specificity towards catechols [26,35].
However, higher activity (4.2 lkatÆkg
)1
) was determined in
extracts of leaf and stem tissues. The moderate rate of
COMT expression in the suspension cells is reminiscent of
the correspondingly low COMT activity in cultured Petro-
selinum crispum cells [36]. In Petroselinum as well as in Ruta
cells, the COMT activity had been clearly distinguished
from the BMT and XMT activities [22,34].
cDNA cloning and functional expression
The total RNA from A. majus cells that had been elicited
for 4 h was used as a template for RT-PCR amplifications,
with degenerate oligonucleotide primers designed for the
cloning of COMT-related enzymes from other plant sources
[24–26]. These experiments generated two different frag-
ments of 215 bp which were cloned into the pCR2.1-TOPO
vector and extended to the full size cDNAs of 1062 and
1074 bp, respectively, by RACE and RLM-RACE (Gen-
Bank accession nos. AY443006 and AY443008). Prelimin-
ary sequence alignments had already revealed a close
similarity of the cDNAs with those of other plant OMTs.
Therefore, the inserts were subcloned into an expression
vector for the expression in E. coli, and the recombinant
polypeptides were extracted from the induced transformants
Fig. 2. Thin-layer cochromatography of the labeled product from BMT
assays with authentic bergaptol and bergapten on silica F
254
plates
developed with n-hexane/ethylacetate/methanol (5 : 5 : 1, v/v/v). Ref-

erence furanocoumarins separated in the absence (lane 1) and in the
presence of the enzymatic product (lane 3) were spotted by their
quenching under irradiation at 254 nm, and the enzymatic product
(lanes 2 and 3) was detected by autoradiography using a Bioimager
(inverse presentation). S, start line; F, solvent front.
Ó FEBS 2004 Bergaptol O-methyltransferase in Ammi majus
1
(Eur. J. Biochem. 271) 935
in 70 m
M
Tris/HCl buffer pH 7.5, containing 10 m
M
EDTA. The enzyme activity of the crude supernatants
was determined with a variety of potential substrates, and
the 1062 bp transformant was found to methylate bergaptol
to bergapten with narrow substrate specificity. The identity
of the enzymatic product was firmly established by thin-
layer cochromatography with authentic bergapten in four
solvent systems (Fig. 2), and hence the transformant
encoded a BMT, designated AmBMT. The functionality
of the 1074 bp clone encoding a COMT-like protein,
however, has so far not been assigned.
Because the RT-PCR from cell culture RNA failed to
amplify a full-size COMT sequence, we turned back to
A. majus plants and used the RNA from leaf and stem
tissues as a template. These experiments yielded a cDNA of
1095 bp (GenBank accession no. AY443007), which was
expressed in E. coli andshowntoencodeaCOMTthat
converts caffeate to ferulate.
Both labeled cDNAs were used as probes for Northern

blotting experiments employing the total RNA of A. majus
cells at various time points following the addition of the
Pmg elicitor. The abundance of BMT transcripts, which
were hardly detectable in control cells, increased signifi-
cantly to a transient maximum at 7 h (Fig. 3). However, a
very weak hybridization signal was recorded for the COMT
transcripts that hardly changes in intensity over the time of
the experiment and corresponded with the low constitutive
level of enzyme activity in the cells.
Characterization of enzymes
The BMT activity ( 2.5 lkatÆkg
)1
) in the desalted bacterial
extracts was very labile and could not be purified exten-
sively, whereas the COMT activity (1.6 lkatÆkg
)1
)remained
stable upon storage. The enzyme extracts were therefore
subjected only to ammonium sulfate fractionation (45–60%
saturation for COMT; 60–80% saturation for BMT) and
subsequent desalting through PD-10 columns, enhancing
the apparent specific activities to 20.2 and 10.3 lkatÆkg
)1
,
respectively, for BMT and COMT. The rates of enzyme
activity were compared in various buffers in pH range
2.0–10.0 and at temperatures ranging from 20 to 50 °C.
Significant activity of the recombinant BMT was observed
between 38 °Cand44°C and from pH 6.5–9.0, and the
optimum was recorded at 42 °C in potassium phosphate

buffer pH 8.0, whereas the optimal COMT activity was
observed at 32 °C in potassium phosphate buffer pH 7.0.
These conditions were routinely chosen for all further
assays. This activity profile of the recombinant BMT was
fully compatible with the data measured for BMT extracted
from A. majus cells (H. Ekiert and R. Lukac
ˇ
in, unpublished
data), and the pH dependency corresponded to that of the
BMTs from P. crispum [22] or R. graveolens [34]. The effect
of a number of metal ions (Co
2+
,Cu
2+
,Fe
2+
,Fe
3+
,
Mg
2+
,Mn
2+
,Ni
2+
,Zn
2+
)at1.5m
M
or 0.1 m

M
concen-
tration was also examined. Significant inhibition of the
BMT activity was observed in the presence of Cu
2+
(100%
Fig. 3. Induction of BMT transcript abundance
in Ammi majus cell cultures. Total RNA (4 lg
per dot) isolated from the cells at different time
intervals following the addition of elicitor
(lanes 1 +, 2 +) or from controls (lanes
3 –, 4 –) treated with water (1 mLÆ40 mL
)1
culture) was employed for Northern dot blot
hybridization using labeled AmBMT cDNA
as a probe.
Table 1. Substrate specificities of A. majus OMTs. Substrates were used at 10 m
M
concentration in the assays. Neither of the OMTs accepted
umbelliferone, psoralen, xanthotoxol, sinapate, 4-coumarate, 2-coumarate, 3-coumarate, catechol, kaempferol, quercetin, dihydrokaempferol,
apigenin or naringenin to a significant extent (< 1%) as a substrate. The relative activity values relate to caffeate as the standard substrate.
Substrate
COMT BMT
Rel. activity (%) K
m
Rel. activity (%) K
m
Bergaptol < 1 100 2.8 l
M
b

Caffeate 100 122.0 l
M
a
<1
5-Hydroxyferulate 177 29.0 l
M
<1
Caffeic acid methyl ester 207 42.0 l
M
<1
Caffeoyl coenzyme A 13.5 219.0 l
M
<1
3-(3,4-dihydroxyphenyl)propionate 10.4 2.2 m
M
<1
Esculetin 27 133.0 l
M
<1
Daphnetin 8 103.0 l
M
<1
a
K
m SAM
¼ 2 l
M
.
b
K

m SAM
¼ 6.5 l
M
.
936 M. Hehmann et al.(Eur. J. Biochem. 271) Ó FEBS 2004
and 51%) and Ni
2+
(47% and 16%) as well as Co
2+
(21%
and 10%). COMT activity was completely inhibited at
either of the Cu
2+
concentrations but less by Ni
2+
(91.5%
and 47%), Mn
2+
(83% and 7.5%), or Co
2+
,Fe
3+
and
Zn
2+
(23–30% and 0–5%). Fe
2+
and Mg
2+
did not affect

the turnover rates. Similar results were reported for
heterologous OMTs [37,38].
A variety of potential substrates, including xanthotoxol
(Table 1), was employed to determine the substrate
specificities of the recombinantly expressed A. majus
BMT and COMT. However, only bergaptol was accepted
as a substrate by the BMT. Kinetic assays revealed the
affinities to S-adenosyl-
L
-methionine and bergaptol
at K
m
¼ 6.5 and 2.8 l
M
, respectively. The COMT was
much less selective and showed the highest affinity to
5-hydroxyferulate (K
m
¼ 29 l
M
) followed by caffeic
acid methyl ester, caffeate, esculetin, caffeoyl-CoA,
3-(3,4-dihydroxyphenyl)propionate (dihydrocaffeate) or
daphnetin (Table 1).
Relationship of sequences
S-Adenosyl-
L
-methionine-dependent O-methyltransferases
are characterized by a common signature of five highly
Fig. 4. Alignment of AmBMT and AmCOMT polypeptides from Ammi majus. Hyphens were inserted for maximal alignment. The consensus

sequence (COMTcons) derived from the COMT polypeptides of Ocimum basilicum, Catharanthus roseus, Capsicum annuum, Capsicum chinense,
Prunus dulcis and Rosa chinensis is given in the bottom line. Identical amino acid residues are denoted by asterisks, and dots mark conservative
exchanges. The amino acids are numbered in the right margin. Highly conserved regions I–V proposed as a signature of S-adenosyl-
L
-methionine-
dependent O-methyltransferases [26,39,40] are underlined, and the motifs 1 and 2 considered to govern the substrate specificity [44,47] are in bold.
Ó FEBS 2004 Bergaptol O-methyltransferase in Ammi majus
1
(Eur. J. Biochem. 271) 937
conserved regions [39–41], and a corresponding consensus
sequence was assigned from plant OMTs [26]. These
elements were also recognized in both the BMT and COMT
sequences from A. majus (Fig. 4; regions I–V) showing
94.5% and 97% identity with the consensus sequence. In
case of rat liver catechol OMT regions I and IV were shown
by X-ray diffraction to be involved in S-adenosyl-
L
-methionine and metal binding [42], and the other three
regions are likely to serve the same purpose. Generally, five
different structural folds have been reported to bind SAM
during catalysis [43], subclassifying the OMTs, but most
plant OMTs, including COMT and the homologous BMT,
belong to class I. In contrast to BMTs, COMTs occur
ubiquituously in plants and have been cloned from many
different sources. Based on the COMTs of Ocimum
basilicum, Catharanthus roseus, Capsicum annuum, Capsi-
cum chinense, Prunus dulcis and Rosa chinensis,whichshare
a sequence identity of about 50% and 72.3% similarity, a
consensus sequence was derived also for the full size
polypeptides (Fig. 4). The AmCOMT sequence was fully

compatible with this consensus sequence showing 57%
identity at 80% similarity. However, in the light of such a
relationship it is particularly notable that the alignment of
the AmBMT polypeptide with the AmCOMT sequence
revealed 64% identity and 78.4% similarity.
Discussion
The activities of XMT and BMT were described previously
from Ruta graveolens [21] and Petroselinum crispum cells
[22]. In case of dark-grown Petroselinum cells both activities
were induced upon elicitor treatment to transient maxima at
30–35 h with PcXMT reaching a three- to fourfold higher
value than PcBMT [22], whereas the constitutive BMT
activity of irradiated Ruta cells far exceeded that of XMT
activity [34]. The native enzymes purified from induced
parsley cells were reported as stable homodimers of 67 kDa
(XMT) and 73 kDa (BMT) being most active in potassium
phosphate buffer of pH 7.5–8.0 (XMT) or of 8.0–8.5
(BMT).ThenativeenzymesfromRuta appeared to be
larger (85 kDa for BMT and 110 kDa for XMT [34]), and
their stability differed greatly in desalted crude extracts.
XMT activity was lost rapidly while the BMT activity
decreased at only a moderate rate [34]. In contrast, BMT
from A. majus was found to be a rather labile enzyme in
crude extract from plant cells or after recombinant expres-
sion, which did not enable the extensive purification.
Furthermore, XMT activity was induced to a negligible
extent in elicited A. majus cells as compared to BMT.
Nevertheless, the expression of AmBMT in E. coli yielded
highly active extracts that revealed a molecular mass
corresponding to that of bovine serum albumin

(67 ± 5 kDa) on size exclusion chromatography calibrated
with alcohol dehydrogenase, bovine serum albumin, oval-
bumin, chymotrypsinogen A and ribonuclease A. Apparent
K
m
values were determined at 2.8 l
M
(bergaptol) and 6.5 l
M
(SAM) which is in accordance with the values reported for
native PcBMT (K
mbergaptol
4.0 l
M
; K
mSAM
3.1 l
M
) [22].
The cloning of AmBMT revealed a molecular mass of
38.7 kDa for the translated polypeptide, strongly suggesting
a homodimer composition for the native Ammi enzyme as
was shown previously for other OMTs [44–46]. The high
degree of homology with heterologous COMTs prompted
us to clone the AmCOMT also, which was achieved using
seedlings. The alignment for these two OMT polypeptides
revealed a surprisingly high degree of homology (Fig. 4).
The similarity to annotated COMTs had been proposed
also for the PcBMT [26], but, unfortunately, the relevant
sequence data have not been published and cannot be

compared. AmBMT and AmCOMT are regulated differ-
ently upon elicitation of A. majus cells, because the low level
of COMT transcript abundance and activity of the cells
hardly changed over the time of the experiments, whereas
the AmBMT transcript was transiently induced to a
maximum at 7 h. More importantly, despite the homology
(Fig. 4) the substrate specificities of the recombinant OMTs
differed greatly. The AmBMT exclusively methylated ber-
gaptol to bergapten (Fig. 1), whereas the AmCOMT
accepted several substrates apart from caffeate, with a
preference for 5-hydroxyferulate (Table 1).
It is thus obvious that small sequence elements, in
addition to the five highly conserved regions required for
SAM binding [39,41], strongly affect the substrate specificity
of OMTs. AmBMT is a typical member of the COMT
family of enzymes which differ from the recently crystallized
small-molecule methyl ester OMTs [45]. In the case of two
crystallized OMTs (chalcone OMT, daidzein 7-OMT) from
Medicago sativa, two such regions of 14 (motif I) and 11
amino acids (motif II) were identified and proposed to
control the specificity [44]. From these and corresponding
sequence elements in COMTs and flavonoid OMTs a
consensus sequence was established (Fig. 4) and used to
predict the substrate specificity of a novel OMT [47]. In
summary, the studies suggested that substitutions of two to
three amino acids in motifs I or II may provide a basis for
the OMT classification, although a reliable prediction was
not possible. This is reminiscent of the few amino acid
substitutions reported for Clarkia OMTs to discriminate
caffeate and (iso)eugenol substrates [48]. On comparison of

AmCOMT and AmBMT only subtle differences in the
motifs I and II were noticed due to five substitutions each
(Fig. 4), most of which were conservative exchanges.
However, the mutation of a single residue at other locations
might considerably shift the specificity of OMTs for related
substrates as has been shown for phenylpropene OMTs
from sweet basil [46]. Future point mutations will reveal the
relevance of these substitutions.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft and
Fonds der Chemischen Industrie is gratefully acknowledged. The
authors are indebted to S. Specker and S. Schreiner for fruitful
discussions, to B. Rohde for chromatography and to A. Batschauer
and O. Panajotow (Pflanzenphysiologie und Photobiologie, Fachb-
ereich Biologie, Universita
¨
t Marburg) for their part in the subcloning
experiments.
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