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

In the work presented here we performed a detailed biochemical characterization of a c-tocopherol methyltransferase c-TMT from Arabidopsis thalianaand of a c-TMT purified from Capsicum an

Characterization of c-tocopherol methyltransferases from

Maria Koch1,2, Rainer Lemke3, Klaus-Peter Heise2and Hans-Peter Mock1

1

Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany;2Albrecht-von-Haller-Institut fu¨r

Pflanzenwissenschaften der Universita¨t Go¨ttingen, Germany;3Sungene 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

thalianaand 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- and c- into b- and a-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 performed with the Arabidopsis 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 nonenzyenzy-mic 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 pathpath-way,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 Synechocystishave 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: mock@ipk-gatersleben.de

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)

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 [14C]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 0.1Mpotassium phosphate buffer

of pH 8 containing 1 mMdithiothreitol and 1 mMEDTA The crude protein extract was desalted through a Sephadex G25 column (200 mL bed volume; Pharmacia Biotech, Freiburg,Germany) against buffer A [50 mM Tris; 1 mM EDTA,3 mMdithiothreitol,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 0–1MNaCl 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.

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 1M

NaCl in buffer B Active fractions were pooled and

subjected to chromatography on a hydroxyapatite column

equilibrated in buffer C [10 mMsodium phosphate,1 mM

EDTA,3 mM dithiothreitol,5% glycerol (v/v),pH 7.3]

Elution of protein was performed by increasing the sodium

phosphate concentration to 400 mM Fractions with c-TMT

activity were further chromatographed on Blue Sepharose

equilibrated in buffer D (buffer solution B with Tris reduced

to 25 mM) After washing,a linear gradient from 0–2M

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 mM potassium phosphate buffer containing 6M

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

a 0.1M potassium phosphate buffer of pH 7 containing

1 mM EDTA and 3 mM 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 mM Na2CO3,56 mM dithiothreitol,2 mM EDTA,2%

(v/v) SDS,12% (w/v) sucrose and 0.25% (w/v)

bromophe-nol blue,incubated for 5 min at 95C and centrifuged in

order to remove insoluble residues Electrophoresis was

according to Laemmli [23] The gels were loaded with either

15 lg or 0.5–3 lg 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 ofCapsicum c-TMT

Radioactive assays with [14C]AdoMet for c-TMT from the

last purification step were performed with 20 lg protein

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 mM NaH2PO4,300 mM NaCl,10 mM imida-zol,800 lg lysozyme; pH 8.0) and subsequent centrifuga-tion at 15 000 and 30 000 g,respectively Purificacentrifuga-tion 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 3.8M (NH4)2SO4 during storage of aliquots prior to subsequent enzyme assays

Assay conditions and analytical methods The assay for the Capsicum enzyme is based on the methylation of exogenous c- into a-tocopherol in the presence of [methyl-14C]AdoMet The reactions were car-ried out for 2 h at 25C in 500 lL medium containing

50 mM Tricine/NaOH (pH 7.5),1 mM MgCl2,50 lM c-tocopherol,25 lM[14C]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 mMTris/HCl (pH 8.5),

25 lM AdoMet,50 lM c-tocopherol,5 mM 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; kex:

295 nm and kem: 332 nm) Elution of tocopherols was isocratically with 100% methanol at a flow rate of

1 mLÆmin)1 Enzyme kinetics The experiments were performed by varying the concentra-tion of substrates in the standard assay and by adding

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 OfficeEXCEL

(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 fromCapsicum 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

was kept at 4C 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 purifipurifi-cation

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)1Æprotein)

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

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.

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 fromCapsicum fruits with the recombinant

c-TMT fromArabidopsis

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 34C (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 Capsicum 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 lg of purified c-TMT After electro-phoresis the gel was stained with Coomassie Brilliant blue.

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- to a-tocopherol and of d- to b-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- and b-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 Kmvalues and Vmaxto Km ratios (Table 2) For the Arabidopsis enzyme the Vmax/Km -quotient was twofold higher for d- than for c-tocopherol Kinetics ofArabidopsis 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)1intercepts 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- and b-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)1proteinÆl 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

[14C]-AdoMet 2.0 ± 0.4 (n ¼ 5) 5.2 ± 1.4 (n ¼ 4) n.d n.d.

Our paper describes the first thorough characterization of

the enzymic properties of c-TMTs from higher plants 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- and d-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.

prenyl residue) of the tocopherol aromatic head group,

recently described by Shintani and DellaPenna [18] and

shown in Fig 1 Calculation of the Vmax/KM ratios for

c- and d-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.

References

1 Wang,X & Quinn,P.J (1999) Vitamin E and its function in membranes Prog Lipid Res 38,309–336.

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Fig 8 Product inhibition kinetics of Arabidopsis c-TMT Hanes–Wilkinson-plots are shown for the product inhibition of c-TMT by AdoHcy (upper panels) and c-tocopherol (lower panels) The set of data correspond to one of six independent experiments All data points are derived from duplicate assays Toc,tocopherol (A1) [c-tocopherol]/v vs [c-tocopherol] at various fixed concentrations of AdoHcy,(A2) [AdoMet]/v vs./ [AdoMet] at various fixed concentrations of AdoHcy (B1) [c-tocopherol]/v vs [c-tocopherol] at various fixed concentrations of a-tocopherol,(B2) [AdoMet]/v vs./[AdoMet] at various fixed concentrations of a-tocopherol.

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