Báo cáo khoa học: Characterization of c-tocopherol methyltransferases from Capsicum annuum L and Arabidopsis thaliana pptx

Characterization of c-tocopherol methyltransferases from
Capsicum annuum
L and
Arabidopsis thaliana
Maria Koch
1,2
, Rainer Lemke
3
, Klaus-Peter Heise
2
and Hans-Peter Mock
1
1
Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany;
2
Albrecht-von-Haller-Institut fu
¨
r
Pflanzenwissenschaften der Universita
¨
tGo
¨
ttingen, Germany;
3
Sungene GmbH & Co. KgaA, Gatersleben, Germany
Tocopherols are essential micronutrients in human and
animal nutrition due to their function as lipophilic anti-
oxidants. They are exclusively synthesized by photosynthetic
organisms including higher plants. Despite the attributed
beneficial health effects and many industrial applications,
research on the tocopherol biosynthetic pathway and its

regulation in plants is still limited. In the work presented here
we performed a detailed biochemical characterization of a
c-tocopherol methyltransferase (c-TMT) from Arabidopsis
thaliana and of a c-TMT purified from Capsicum annuum
fruits, a tissue with high accumulation of tocopherols. The
biochemical characteristics of both enzyme preparations
were remarkably similar including substrate specificities.
Both enzymes converted d-andc-intob-anda-tocopherol,
respectively, but b-tocopherol was not accepted as a sub-
strate, pointing to a specific methylation at the C(5)-position
of the tocopherol aromatic head group. A kinetic analysis
performedwiththeArabidopsis enzyme was consistent with
an iso-ordered bi-bi type reaction mechanism. Our results
emphasize the role of c-TMT in regulating the spectrum of
accumulated tocopherols in plants.
Keywords: Arabidopsis; Capsicum; c-tocopherol; methyl-
transferase; vitamin E.
a-Tocopherol belongs to a family of lipid-soluble hydrocar-
bon compounds characterized by a chromanol ring with a
phytyl side chain and summarized under the collective name
Vitamin E. Putative biochemical functions of these com-
pounds are the antioxidant properties as efficient scavengers
of lipid peroxyl radicals and their action as membrane
stabilizers [1]. Tocopherols have been found in all green
tissues of photosynthetic organisms [2], but significant
amounts are frequently observed in seeds. Plant tissues
highly active in photosynthesis bear a great potential for the
generation of reactive oxygen species and chloroplasts
possess an elaborated protective system composed of enzy-
mic and nonenzymic components [3]. It is assumed that the

lipophilic tocopherols complement the antioxidative func-
tion of the hydrophilic ascorbate in a concerted manner [4].
Besides their functions in plant metabolism, tocopherols
are essential components of the human diet and serve as
protectants in food and pharmaceutical technology [5].
Understanding the biochemical pathway of tocopherol
biosynthesis therefore opens the perspective for improving
the nutritional quality of crop plants [6]. Biosynthesis of
tocopherols was demonstrated in plastid envelopes [7] from
precursors originating from the plastidial isoprenoid path-
way and from the shikimate pathway, providing the
hydrophobic phytyl moiety and the polar head group
homogentisic acid, respectively. Furthermore, plastidial
tocopherol accumulation appears to depend on the
up-regulation of genes encoding the enzymes being involved
in the formation of these precursors, like 1-deoxyxylulose
5-phosphate synthase [8], geranylgeranyl reductase [9] and
4-hydroxyphenylpyruvate dioxygenase [10]. Based on earlier
investigations [11] and on detailed work on the chemical
synthesis of prenylquinones [12] the pathway for plastidial
a-tocopherol biosynthesis has been elucidated [13,14]. The
proposed pathway includes the phytylation of homogentisic
acid to form 2-methyl-6-phytylquinol, the first ring methy-
lation at position 3 to yield 2,3-dimethyl-5-phytylquinol,
cyclization to yield c-tocopherol, and finally a second ring
methylation at position 5 to yield a-tocopherol (Fig. 1).
Detailed biochemical analysis of tocopherol synthesis and
its regulation has largely been hampered by the lack of
purified enzyme preparations catalysing individual steps of
the pathway. Earlier reports have focused on the purifica-

tion of c-TMT from bell pepper (Capsicum annuum)fruits
[15], from spinach [16] and Euglena [17]. Consistent with
previous tracer experiments these studies have shown that
the c-TMT activities were membrane-associated and had to
be solubilized prior to any additional purification step. A
purified c-TMT enzyme preparation was reported for bell
pepper indicating a molecular mass of 33 kDa for the active
monomeric form [15]. Due to the instability of the
solubilized enzyme, purification to homogeneity was not
reported for the Euglena and spinach enzyme preparations.
Recently genes encoding c-TMTs from Arabidopsis and
Synechocystis have been identified [18]. Overexpression of
the Arabidopsis enzyme with a seed-specific promoter
resulted in a more than 80-fold increase of a-tocopherol at
Correspondence to H P. Mock, Institute of Plant Genetics and
Crop Plant Research, Corrensstrasse 3, D-06466 Gatersleben,
Fax: + 49 39482 5139, Tel.: + 49 39482 5506,
E mail:
Abbreviations: AdoHcy, S-adenosyl-
L
-homocystein; AdoMet,
S-adenosyl-
L
-methionine; c-TMT, c-tocopherol methyltransferase;
toc, tocopherol.
Enzymes: c-tocopherol methyltransferase (EC 2.1.1.95); accession
number AF104220.
(Received 23 July 2002, revised 3 October 2002,
accepted 14 November 2002)
Eur. J. Biochem. 270, 84–92 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03364.x

the expense of c-tocopherol without changing the total
content. The recombinant enzyme expressed in E. coli
accepted d-, but not b-tocopherol in addition to c-toco-
pherol as a substrate. In the present paper we attempted a
detailed characterization of c-TMT activities with respect to
kinetic properties and substrate specificities. We investigated
the properties of the recombinant enzyme from Arabidopsis
and of a partially purified preparation from bell pepper fruit
pericarp to compare the characteristics of c-TMT enzymes
from different species and tissues. For purification of
c-TMT we have chosen the fruit pericarp of Capsicum
which is a tissue with a high enrichment of tocopherols.
Materials and methods
Plant material
Mature Capsicum annuum L. fruits of the red variety were
obtained from a local market.
Chemicals
The (+) c-and(+) d-tocopherols were purchased from Sigma
(Deisenhofen, Germany). Residual (+/–)-b-tocopherols
were obtained from Merck (Darmstadt, Germany) and
additionally checked for purity by HPLC. [
14
C]AdoMet
(1.85 MBq) was from Pharmacia Biotech (Freiburg, Ger-
many) and unlabelled AdoMet and AdoHcy were from
Sigma. Chromatographic materials and columns were
obtained from Bio-Rad (hydroxyapatite), Phenomenex
(BioSep–Sec-S3000) and Pharmacia (all others).
All other chemicals were of analytical grade and obtained
from various suppliers.

Preparation and purification of c-TMT
from pepper fruits
Chromoplast membranes were isolated from 12 kg of fruit
pericarp as described by Arango and Heise [19–21] and
precipitated with acetone according to d’Harlingue and
Camara [15]. Solubilization of c-TMT was performed as
described [19–21] using 0.1% (w/v) Tween 80 as a detergent.
The resulting crude protein extract was either used for the
characterization of c-TMT activities or further precipitated
by sequential saturation (20–50%) with ammonium sul-
phate and redissolved in 0.1
M
potassium phosphate buffer
of pH 8 containing 1 m
M
dithiothreitol and 1 m
M
EDTA.
The crude protein extract was desalted through a Sephadex
G25 column (200 mL bed volume; Pharmacia Biotech,
Freiburg, Germany) against buffer A [50 m
M
Tris; 1 m
M
EDTA, 3 m
M
dithiothreitol, 3% (v/v) glycerol; pH 7.2] and
purified by subsequent chromatography (FPLC system;
Pharmacia Biotech, Freiburg, Germany) starting with a
DEAE-Sepharose (fast flow material) column of 200 mL

equilibrated in buffer A. After removal of nonbound
proteins, elution of c-TMT was performed with a linear
gradient from 0–1
M
NaCl in buffer A. Fractions containing
c-TMT activity were concentrated and applied to a second
Fig. 1. Proposed pathway for the biosynthesis of tocopherols in plants.
Ó FEBS 2003 c-tocopherol methyltransferases (Eur. J. Biochem. 270)85
DEAE-Sepharose column (20 mL bed volume) equilibrated
in buffer B (buffer A adjusted to pH 7.8). After washing the
column was developed with a linear gradient up to 1
M
NaCl in buffer B. Active fractions were pooled and
subjected to chromatography on a hydroxyapatite column
equilibrated in buffer C [10 m
M
sodium phosphate, 1 m
M
EDTA, 3 m
M
dithiothreitol, 5% glycerol (v/v), pH 7.3].
Elution of protein was performed by increasing the sodium
phosphate concentration to 400 m
M
. Fractions with c-TMT
activity were further chromatographed on Blue Sepharose
equilibrated in buffer D (buffer solution B with Tris reduced
to 25 m
M
). After washing, a linear gradient from 0–2

M
NaCl in buffer D was applied to elute bound proteins.
Between column separations the active fractions were
desalted on Sephadex G25 or pooled and concentrated by
dialysis against polyethyleneglycol 35 000 (Merck, Darms-
tadt, Germany). Other methods for enzyme concentration
such as ultrafiltration led to considerable losses of enzyme
activity presumably due to the unspecific binding of the
hydrophobic protein. Further purification of the concen-
trated labile protein was attempted by precipitation with
chloroform/methanol according to Wessel and Flu
¨
gge [22]
and subsequent separation by HPLC under denaturing
conditions on a BioSep–Sec-S3000 (300 · 7.8 mm) gel
filtration column (Phenomenex, Aschaffenburg, Germany)
using 20 m
M
potassium phosphate buffer containing 6
M
guanidine hydrochloride.
Molecular mass determination
The native molecular mass was determined by gel filtra-
tion on a Superdex 200 HR 30/10 column (1 · 30 cm) with
a0.1
M
potassium phosphate buffer of pH 7 containing
1m
M
EDTA and 3 m

M
dithiothreitol at a flow rate of
0.5 mLÆmin
)1
. Fractions of 1.25 mL were collected. Col-
umn calibration was with a protein standard containing
aldolase (160 kDa), BSA (68 kDa), ovalbumin (45 kDa),
carboanhydrase (30 kDa) and myoglobin (17.8 kDa).
SDS polyacrylamide gel electrophoresis
The samples were dissolved in a buffer medium containing
56 m
M
Na
2
CO
3
, 56 m
M
dithiothreitol, 2 m
M
EDTA, 2%
(v/v) SDS, 12% (w/v) sucrose and 0.25% (w/v) bromophe-
nol blue, incubated for 5 min at 95 °C and centrifuged in
order to remove insoluble residues. Electrophoresis was
according to Laemmli [23]. The gels were loaded with either
15 lgor0.5–3lg protein, electrophoresed and stained with
Coomassie blue or silver according to Jungblut and Seifert
[24]. Protein markers were from the LMW calibration kit of
Pharmacia Biotech (Freiburg, Germany).
Protein determination

Protein was measured according to Bradford [25] using the
reagent solution from Bio-Rad (Munich, Germany) and
BSA as standard protein.
Photolabelling of
Capsicum
c-TMT
Radioactive assays with [
14
C]AdoMet for c-TMT from the
last purification step were performed with 20 lgprotein
under UV-irradiation for 2 h according to Subbaramaiah
and Simms [26]. The protein was precipitated and
re-dissolved as described by Wessels and Flu
¨
gge [22] and
separated by SDS/PAGE. Radioactively labelled proteins
were visualized using a Phosphoimaging system (Storm
system; Amersham Biotech, Freiburg, Germany).
Purification of the recombinant c-TMT from
Arabidopsis thaliana
An E. coli strain for overexpressing Arabidopsis c-TMT [18]
was a generous gift of SunGene GmbH & Co. KGaA
company, Gatersleben, Germany. After harvesting the
induced cells the recombinant protein was released by
ultrasonication (6 · 15 s) of the cells in an ice-cold buffer
medium (50 m
M
NaH
2
PO

4
, 300 m
M
NaCl, 10 m
M
imida-
zol, 800 lg lysozyme; pH 8.0) and subsequent centrifuga-
tion at 15 000 and 30 000 g, respectively. Purification was
performed using an FPLC system on a Ni-agarose column
(5–10 mg protein per ml Qiagen Ni-NTA Superflow;
1 · 10 cm; flow rate: 0.5 mLÆmin
)1
; 10 mL fractions) by
stepwise elution with increasing imidazol concentrations in
the buffer medium according to the manufacturer’s proto-
col. The enzyme activity was preserved by additions of
10–20% glycerol or 3.8
M
(NH
4
)
2
SO
4
during storage of
aliquots prior to subsequent enzyme assays.
Assay conditions and analytical methods
The assay for the Capsicum enzymeisbasedonthe
methylation of exogenous c-intoa-tocopherol in the
presence of [methyl-

14
C]AdoMet. The reactions were car-
ried out for 2 h at 25 °Cin500lL medium containing
50 m
M
Tricine/NaOH (pH 7.5), 1 m
M
MgCl
2
, 50 l
M
c-tocopherol, 25 l
M
[
14
C]AdoMet and 0.1–0.7 mg protein.
c-Tocopherol or other tocopherols used as substrate were
added from concentrated stock solutions in ethanol into the
enzyme assays. The reaction products were extracted
according to Arango and Heise [19,20] and separated on
HPTLC-silicagel 60 plates (Merck, Darmstadt, FRG) with
toluene as the solvent. The product formation was moni-
tored using a Phosphoimager system (Storm system;
Amersham Biotech, Freiburg, Germany).
The recombinant enzyme was measured in a modified
nonradioactive assay containing 50 m
M
Tris/HCl (pH 8.5),
25 l
M

AdoMet, 50 l
M
c-tocopherol, 5 m
M
dithiothreitol
and 1–5 lg of the purified enzyme protein in a total volume
of 500 lL. After termination the assay was processed as
described above except that the residues of the organic
phase were dissolved in methanol. The a-tocopherol content
was quantified after HPLC (Waters 2690 Separation
Module) separation on a Prontosil 200–3-C30-column
(Bischoff Chromatography; NC; 230 · 4.6 mm, 3.0 lm)
by fluorescence detection (Jasco FP-920 detector; k
ex
:
295 nm and k
em
: 332 nm). Elution of tocopherols was
isocratically with 100% methanol at a flow rate of
1mLÆmin
)1
.
Enzyme kinetics
The experiments were performed by varying the concentra-
tion of substrates in the standard assay and by adding
86 M. Koch et al.(Eur. J. Biochem. 270) Ó FEBS 2003
appropriate amounts of inhibitors (product inhibition
experiments). Details are given in the individual experiments
in the results section. Data were analyzed by linear
regression using the statistic program of MS Office

EXCEL
(Microsoft, Deisenhofen, Germany).
Statistics
Substrate interaction kinetic experiments and product
inhibition reactions were performed at least six times. All
other experiments were conducted at least three times.
Results
Purification of c-TMT activity from
Capsicum annuum
fruits
A crude protein extract was prepared by acetone precipi-
tation from red bell pepper fruits characterized by the
highest specific c-TMT activity when compared with other
fruit varieties [27]. The crude protein extract could be stored
at )20 °C without loss of c-TMT activity for 4 weeks, but
approximately half of the activity was lost when the extract
waskeptat4°C for 5 days. After solubilization and
ammonium sulphate precipitation (saturation up to 50%)
the crude c-TMT was further enriched by anion exchange
(twice) followed by chromatography on hydroxyapatite and
Blue Sepharose (Table 1). Additional gel filtration of the
native enzyme on Superdex 200 showed no further purifi-
cation effect. In total an approximately 45-fold purification
with a 9% recovery of c-TMT activity was achieved. The
enzyme purification during the subsequent steps was
assessed by SDS gel electrophoreses as shown in Fig. 2
demonstrating the effectiveness of individual purification
steps. All attempts to further enrich the native c-TMT by
for example anion exchange chromatography or affinity
chromatography on Adenosine-Sepharose in order to

obtain an apparently pure fraction were hampered by the
loss of enzyme activity. Addition of detergents into the
buffer solutions did not stabilize enzyme activity (data not
shown). Chromatography on reversed phase material led to
severe loss of protein presumably by interactions with the
gel matrix (data not shown). Further separation of proteins
contained in the most purified active enzyme fraction was
only achieved under denaturing conditions by HPLC on
BioSep–Sec-S3000 (manufacturer) according to their mo-
lecular size, but protein amounts were not sufficient for
sequencing of candidate protein bands with a molecular
mass predicted from gel filtration and photoaffinity labelling
(data not shown). Addition of divalent cations, BSA and
yolk lipids had no protective influence on the stability of the
enzyme (data not shown).
Molecular mass determination
Gel filtration (Fig. 3) and photoaffinity labelling followed
by SDS/PAGE were used to determine the molecular mass
of c-TMT (Fig. 4). When applying the crude protein extract
obtained after acetone precipitation to a Superdex 200 gel
filtration column, c-TMT activity eluted in the high
molecular mass fraction with a native molecular mass of
more than 600 kDa. In contrast a mass of approximately
36 kDa was observed when the most purified fractions after
affinity chromatography were analyzed (Fig. 3). It was
tentatively concluded that this mass would represent the
monomeric state of the enzyme. To further corroborate this
assumption we used photoaffinity labelling and SDS/PAGE
as an additional method for molecular mass determin-
ation. For photoaffinity labelling of c-TMT a fraction after

Blue Sepharose column purification was used. During the
enzyme assay UV light (254 nm) was applied to enable the
eventual covalent binding of radioactively labelled substrate
to a fraction of the c-TMT as already demonstrated for
other methyltransferases [26–28]. After termination of the
Table 1. Purification protocol of c-TMT from red Capsicum fruits.
Fraction
Volume
(mL)
Total protein
(mg)
Total activity
(fkat)
Specific activity
(fkatÆmg
)1
Æprotein)
Recovery
(%)
Purification
(fold)
Chromoplast membranes 500 2103 18164 9.5 100 1
Acetone precipitate 98 1161 20501 17.7 102 2
50% (NH
4
)
2
SO
4
54 610 24086 37.0 113 4

DEAE (I) 110 187 15481 82.8 85 9
DEAE (II) 54 70 10834 154.1 60 16
Hydroxyapatite 48 19 4541 235.4 25 25
Blue Sepharose 92 4 1647 426.8 9 45
Fig. 2. SDS/PAGE analysis of fractions obtained during subsequent
steps of c-TMT purification from Capsicum fruits. From each step, 1 lg
of protein was loaded on the gel. Proteins were visualized by silver
staining. The following abbreviations are used for labelling of the
lanes: CM, chromoplast membrane; AE, acetone precipitate, AS,
ammonium sulphate precipitate; IE I + II, active fractions from
subsequent DEAE sepharose columns; HA, active fractions eluted
from the hydroxyapatite column; AF, active fractions obtained after
chromatography on Blue Sepharose; M, molecular mass marker.
Ó FEBS 2003 c-tocopherol methyltransferases (Eur. J. Biochem. 270)87
reaction, proteins were separated on SDS gel electrophor-
esis. Imaging analysis revealed the presence of one single
labelled protein band with a molecular mass of approxi-
mately 36 kDa (Fig. 4A).
Comparison of the properties of the partially purified
c-TMT from
Capsicum
fruits with the recombinant
c-TMT from
Arabidopsis
Recombinant Arabidopsis c-TMT containing a His-tag was
purified from E. coli cell lysates by affinity chromatography.
Analysis of the purified fraction by SDS/PAGE showed a
single band with the expected molecular mass (Fig. 4B).
Like the c-TMT from Capsicum fruits the Arabidopsis
enzyme was slightly stimulated by dithiothreitol and was

not dependent on divalent cations (data not shown).
The pH dependence of both c-TMT sources was
evaluated in the range of 5.5–10.0 with different buffer
systems (Fig. 5) as described under materials and methods.
The recombinant c-TMT from A. thaliana showed a more
alkaline and sharper pH-optimum at pH 8.5 than the
partially purified enzyme from pepper pericarp which
showed a broader curve with a maximum at pH 7.5.
Stability tests by preincubating the Arabidopsis enzyme at
different pH values followed by assaying the activity at 8.5
indicated that the sharp decline of activity towards lower pH
values was only partially due to enzyme inactivation (data
not shown).
For both enzyme preparations the methyltransferase
reaction showed an identical temperature maximum of
approximately 34 °C (data not shown).
Substrate specificities
To elucidate putative differences in the molecular properties
of both enzymes, a detailed investigation of their substrate
specificities was performed with the recombinant Arabidop-
sis enzyme and a c-TMT fraction from Capsicum fruits
obtained by solubilizing the acetone precipitate. Both
c-TMT preparations were incubated with different
Fig. 3. Elution profile of c-TMT on a Super-
dex-200 gel filtration column. The insert shows
the calibration curve obtained by using
standard proteins (aldolase, 160 kDa; bovine
serum albumin, 68 kDa; ovalbumin, 45 kDa;
carboanhydrase, 30 kDa; myoglobin
17.8 kDa). (s), enzyme activity; (n), protein.

Fig. 4. Photoaffinity labelling of Ca psicum
c-TMT band from a Blue Sepharose column
fraction (A) and SDS/PAGE analysis of puri-
fied recombinant Arabidopsis c-TMT (B).
(A) The protein extract (20 lg) was incubated
with 14 l
M
[
14
C]AdoMet under UV-irradi-
ation. After termination of the assay the pro-
tein fraction was separated by SDS/PAGE.
Radioactively labelled proteins were visualized
by phosphoimaging. (B) SDS/PAGE of
recombinant c-TMT from A. thaliana purified
by chromatography on Ni-agarose, loaded
with 5 lgofpurifiedc-TMT. After electro-
phoresis the gel was stained with Coomassie
Brilliant blue.
88 M. Koch et al.(Eur. J. Biochem. 270) Ó FEBS 2003
methyl-substituted tocopherols. In the case of c-TMT from
pepper the labelled reaction products were separated by
HPTLC and visualized by phosphoimaging (Fig. 6). The
unlabelled reaction products after incubation of the Ara-
bidopsis enzyme were separated by HPLC and detected
fluorimetrically. Both enzyme preparations showed the
conversion of c-toa-tocopherol and of d-tob-tocopherol,
respectively, whereas b-tocopherol was not accepted as
substrate (Fig. 6; Table 2). The acceptance of c-tocopherol
and of d-tocopherol by the crude TMT preparation was also

kept through all the subsequent purification steps (data not
shown). These results indicated that the final methylation
step leading to the formation of a-andb-tocopherol,
respectively, is exerted by one enzyme.
Kinetic properties
The properties of both enzyme preparations were further
compared by a thorough analysis of kinetic parameters. The
c-TMT activities from both sources showed regular Micha-
elis–Menten behaviour for all substrates tested (data not
shown). For all substrates investigated, the pepper c-TMT
preparation showed very similar K
m
values and V
max
to K
m
ratios (Table 2). For the Arabidopsis enzyme the V
max
/K
m
-
quotient was twofold higher for d- than for c-tocopherol.
Kinetics of
Arabidopsis
TMT
The following kinetic analysis was performed for the
forward reaction in the presence and absence of inhibitors.
Substrate interaction kinetic experiments were performed
by varying one substrate at different fixed concentrations of
the other substrate (Fig. 7). When either c-tocopherol or

AdoMet were varied double reciprocal plots yielded lines
converging to the left of the ordinate axis. Secondary plots
of V
)1
intercepts and of slopes were linear for either of the
substrates. These results indicated that the c-TMT methy-
lation reaction follows a sequential reaction mechanism and
are accordingly not consistent with a ping-pong mechanism.
Product inhibition studies
In order to discriminate between the possible kinetic
mechanisms suggested by the initial velocity studies, product
inhibition experiments were performed with either one of
the products of the reaction, a-tocopherol or AdoHcy
(Fig. 8).
Variation of both c-tocopherol or AdoMet as substrates
in the presence of either a-tocopherol or AdoHcy as
inhibitors always led to a noncompetitive inhibition (Fig. 8).
This pattern of product inhibition is consistent by assuming
that the methylation reaction follows an iso-ordered bi-bi
mechanism.
Fig. 6. Substrate specificity of partially purified TMT from Capsicum.
Enzyme assays were performed with [
14
C]AdoMet in the presence of
d-tocopherol (lane A) or c-tocopherol (lane B). Reaction products
were separated by HPTLC and visualized by phosphoimaging. Prod-
uct formation was verified by cochromatography with nonlabelled
a-andb-tocopherol standards, which were detected by their fluores-
cence under UV-light. In a control reaction, tocopherol was omitted as
a substrate.

Fig. 5. Influence of pH on c-TMT activity. Assays were performed at
different pH values in the following buffers: Mes, pH 5.5–6.5; potas-
sium phosphate, pH 6.5–8.0; Tris/HCl, pH 7.5–9.0; carbonate buffer,
pH 9.2–10.0; (r), partially purified c-TMT from Capsicum;(d) Ara-
bidopsis c-TMT.
Table 2. Kinetic parameters of c-TMT partially purified from Capsicum fruits and of a recombinant c-TMT (A. thaliana ). Data sets were evaluated
according to the method of Hanes–Wilkinson; n.d., not determined.
Substrate
K
m
[l
M
] V
max
/K
m
[fkatÆmg
)1
proteinÆl
M
)1
]
Capsicum Arabidopsis Capsicum Arabidopsis
c-Tocopherol 3.1 ± 0.5 (n ¼ 6) 5.4 ± 0.6 (n ¼ 4) 15.8 2700
d-Tocopherol 2.9 ± 0.7 (n ¼ 5) 3.3 ± 0.5 (n ¼ 4) 12.8 6500
b-Tocopherol no product no product – –
[
14
C]-AdoMet 2.0 ± 0.4 (n ¼ 5) 5.2 ± 1.4 (n ¼ 4) n.d. n.d.
Ó FEBS 2003 c-tocopherol methyltransferases (Eur. J. Biochem. 270)89

Discussion
Our paper describes the first thorough characterization of
the enzymic properties of c-TMTsfromhigherplants.The
present purification protocol for c-TMT from pepper fruits
was initially based on a previously published scheme [15].
Despite several modifications we were not able to purify
c-TMT to complete homogeneity (Fig. 2) although the
purification factor of 45 was similar to previously published
results [15]. Analysis of the most enriched fraction by SDS/
PAGE and sensitive silver staining revealed the presence of
a range of bands. A faint band at the expected molecular
mass of 36 kDa was visible but obviously representing only
a small portion of the total protein content of this fraction.
To this end it remains unclear how c-TMT from pepper
fruits could be purified to apparent homogeneity by a
69-fold enrichment starting from a crude membrane
preparation as described in a previous publication [15].
The relatively high native molecular mass of more than
600 kDa, estimated for the crude pepper c-TMT after the
first purification steps resembles the earlier findings of
d’Harlingue and Camara [15] and may be, indeed, due to
the tendency of membrane proteins to aggregate. This
aggregation phenomenon may also explain the instability of
the membrane enzyme in the diluted state and the high loss
of enzyme activities during the subsequent purification
procedure. In contrast, gel filtration of the purified protein
at the end of the conventional purification procedure
suggests a native molecular mass for the c-TMT from
pepper pericarp of approximately 36 kDa (Fig. 3). This
molecular mass for the monomer is supported by photoaf-

finity labelling [28,29] of the pepper enzyme after SDS-gel
electrophoresis of the Blue Sepharose column fraction
(Fig. 4A) and agrees with the molecular mass of c-TMT
from A. thaliana (Fig. 4B). The presence of only one
labelled band is also indicative that the highly aggregated
form of c-TMT observed during initial steps of purification
contains only one protein involved in methylation of
tocopherol. The low protein amount of the 36 kDa-band
from pepper (Fig. 2) was not sufficient to obtain sequence
information by EDMAN degradation. The 200-fold higher
specific activity of the Arabidopsis c-TMT compared with
the partially purified pepper enzyme also reflects the degree
of purity as well as the loss of activity during lengthy
conventional protein purification procedures.
In spite of significant differences in the purification degree
of the c-TMTs from Capsicum and Arabidopsis, both
enzyme sources show remarkable conformities with respect
to temperature maxima and pH-optima (Fig. 5), substrate
specificities and kinetic parameters (Fig. 3; Table 2). Our
data are consistent with the selected parameters from
previously published initial studies on c-TMT from pepper
and Euglena [15–18]. Both enzyme preparations accepted
c-andd-tocopherol, but not b-tocopherol as a substrate.
This observation points to the specific methylation by this
enzyme at the C(5)-position (i.e. in ortho-position to the
Fig. 7. Substrate interaction kinetics of Arabidopsis c-TMT. Left panels: Lineweaver–Burk-plots for the two-substrate reaction of c-TMT with (A)
1/v against 1/[AdoMet] with c-tocopherol at various fixed concentrations and (B) 1/v against 1/[c-tocopherol] with AdoMet at various fixed
concentrations. Right panels: slope and intercept replots corresponding to A (upper two) or B (lower two) on the left panel.
90 M. Koch et al.(Eur. J. Biochem. 270) Ó FEBS 2003
prenyl residue) of the tocopherol aromatic head group,

recently described by Shintani and DellaPenna [18] and
shown in Fig. 1. Calculation of the V
max
/K
M
ratios for
c-andd-tocopherol showed similar values for the pepper
enzyme. For Arabidopsis c-TMT a more than twofold
higher value was deduced for d-tocopherol relative to
c-tocopherol indicating a higher catalytic efficiency for this
substrate.
Initial velocity experiments in the absence of inhibitors
with variable concentrations of c-tocopherol or AdoMet
(Fig. 7) suggested that the methylation reaction follows a
sequential and not a ping-pong type of reaction mechanism.
In the product inhibition studies all substrate and inhibitor
combinations investigated resulted in a noncompetitive
inhibition pattern (Fig. 8) which is consistent with an iso-
ordered bi-bi mechanism of the methylation reaction. The
mechanism is a special case of the ordered bi-bi mechanism,
which is a consequence of an isomerization of the enzyme in
the central complex [30]. Kinetic analysis of methyltrans-
ferases have revealed sequential as well as ping-pong
mechanisms [31–33]. Two closely related methyltransferases
involved in the biosynthesis of isoquinoline alkaloids
displayed different types of reaction mechanisms [31].
Experimental techniques such as presteady state kinetic
analysis, isotope–partitioning experiments and the use of
mutants were applied to explore the kinetic and catalytic
properties of methyltransferase reactions in more detail [33]

and will help to further define the reaction mechanism of
c-TMT.
It has been recently shown that seed-specific overexpres-
sion of a homogentisate phytyl transferase led to increased
tocopherol levels in transgenic Arabidopsis lines [34] whereas
overexpression of c-TMT resulted in a shift from c-to
a-tocopherol [18]. As individual tocopherols have different
properties, a detailed characterization of further enzymic
steps in the tocopherol biosynthetic pathway such as shown
here for c-TMT will be fundamental to support the rational
design of engineered crop plants with modified profiles of
tocopherols. Interplay between already known proteins and
yet unknown factors will be elucidated by protein interac-
tion studies using approaches such as the yeast two-hybrid
system or pull-down assays. Analysis of transgenic lines and
mutants with modified activities of individual components
such as c-TMT will enable the study of the regulatory
processes of the tocopherol biosynthetic pathway in planta.
Acknowledgements
This work was supported by grants of the SunGene GmbH & Co.
KGaA company, Gatersleben, Germany to M.K., K P.H. and H P.M.
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