Partial Purification and Characterization of S-Adenosyl-L-Methionine: Norreticuline N-Methyltransferases from Berberis Cell Suspension Cultures*

Chi-Kit Wat*, Paul Steffens, and Meinhart H. Zenk Lehrstuhl Pharmazeutische Biologie der Universität München Z. Naturforsch. 41c, 126—134 (1986); received July 3, 1985 Dedicated to Professor Hans Grisebach on the occasion of his 60th birthday

Isoquinoline Alkaloids, Norreticuline, Reticuline, Biosynthesis, Plant Cell Culture Two new N-methyltransferases (NMT-I and NMT-II) were found to occur in Berberis vulgaris cell suspension cultures. One of these enzymes (NMT-I) was partially purified (100-fold) and characterized. This enzyme is specific for tetrahydrobenzylisoquinoline alkaloids and S-adenosyl- L-methionine serves as the methyl donor. The apparent molecular weight of the enzyme is 68,000. The pH optimum of the enzyme is 7.6, the temperature optimum 35 °C. Apparent KM values for (/?)-tetrahydropapaverin as substrate were 0.2 m M and for SAM 0.04 m M . The preparation of the same type of enzyme from B. wilsoniae var. subcaulialata was utilized as an efficient enzymatic system for the synthesis of stereochemically pure (/?)- as well as (S)-reticuline labelled with tritium or 14C at the N —CH3 group. Enzymes catalyzing this type of reactions are named S-adenosyl-L- methionine: norreticuline N-methyltransferases.

Introduction generally assumed that O-methylation precedes Reticuline, a tetrahydrobenzylisoquinoline alka­ N-methylation [4, 5] and our recent experimental loid, is firmly established as the key intermediate in evidence using cell-free extracts verifies this assump­ the biosynthesis of a vast number of isoquinoline tion. In the biosynthesis of orientaline, for instance, alkaloids [1]. Among this group of alkaloids are com­ it has been clearly shown that 6-O-methylnorlau- pounds with such diverse types of structures as danosoline is exclusively monomethylated at position pavines, dibenzopyrrocolines, morphinans, apor- 5' by a highly specific enzyme to yield nororientaline phines, protoberberines, benzophenanthridines and [6], While another enzyme specific for the methyla­ phthalide isoquinolines. Investigating the early steps tion of 6-O-methylnorlaudanosoline at the 4' posi­ in the reticuline pathway, we succeeded in the isola­ tion to give norreticuline is presently under investiga­ tion and partial purification of two new enzymes: tion in our group, we report here the isolation of (S)-norlaudanosoline synthase, the first enzyme in N-methyltransferases which show specificity towards the benzylisoquinoline pathway which stereospecifi- certain benzylisoquinoline alkaloids and which are cally condenses dopamine with 3,4-dihydroxyphenyl- absolutely inactive towards simple biogenic amines. acetaldehyde and a specific O-methyltransferase This N-methylation is the final reaction involved in which methylates norlaudanosoline predominantly at the early steps of the tetrahydroisoquinoline path­ the 6-position yielding 6-O-methyl-norlaudanosoline way, that is, the formation of reticuline. The pres­ [2, 3]. Two further methylation steps, one O-methy- ence of N-methyltransferases had to be postulated, lation at 4' position and one N-methylation at atom since it was shown previously that the latex of 2, will transform this product into reticuline. It is Papaver somniferum, when incubated with nor­ laudanosoline and 14C-SAM, yielded labelled reti­ * Present address: C.-K. Wat, University of British Co­ culine as well as morphinan alkaloids [7], and the lumbia, Department of Botany, Vancouver 8, Canada. same type of experiment with a cell-free extract from Abbreviations: BSA, bovine serum albumin; NLS, nor­ agar-grown callus of Berberis aggregata yielded label­ laudanosoline; NMT, N-methyltransferase; NRK, nor­ led protoberberine alkaloids [8]. Furthermore, in reticuline; OMT, O-methyltransferase; SAM, S-adenosyl- L-methionine; THP, tetrahydropapaverine. this latter study [8], it was demonstrated that tetra­ Reprint requests to M. H. Zenk, Karlstraße 29, D-8000 hydropapaverine was N-methylated. We describe München 2. here the detection, partial purification and charac­ Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen terization of a norreticuline N-methyltransferase 0341 -0382/86/0100 - 0126 $01.30/0 which is of considerable importance in the biosyn- Chi-Kit Wat et al. ■ Norreticuline N-Methyltransferases 127 thesis of the numerous isoquinoline alkaloids and menine-HCl were samples from Prof. E. Broch- which can conveniently be used to introduce radio­ mann-Hanssen, San Francisco; norglaucine, norbol- active or stable isotopes into this important pre­ dine, and caaverine from Prof. Cave, Chatenay- cursor. Malabry; ( R )- and (5)-reticuline and (i?,S)-nor- reticuline from Prof. N. Nagakura, Kobe; ( R,S)- laudanidine from Merck, Darmstadt, and laudano- Materials and Methods sine from Aldrich Chem. Co. (/?,S)-Tetrahydro- Plant material papaverine-HCl was prepared by the method de­ scribed in [10] and resolved into each enantiomer as Berberis vulgaris and B. wilsoniae var. subcaulia- described in [11], (/?,S)-Norlaudanosoline was syn­ lata as well as other Berberis cell cultures used were thesized by a Pictet-Spengler condensation of initiated and maintained since 1975 on Linsmaier and dopamine and 3,4-dihydroxyphenylacetaldehyde [2] Skoog (LS) medium [9]. Batch cultures in 1-litre in water at pH 4. Erlenmeyer flasks containing 250 ml medium were agitated on a gyratory shaker (100 rpm) in diffuse light (750 lux) at 24 °C and were subcultured at Assay methods weekly intervals using about 10% inoculum (v/v). Three substrates were used for assaying the en­ The cells were harvested immediately after reaching zymes. THP and NRK were used to test for the N- stationary phase, frozen in liquid nitrogen and stored methyltransferases and caffeic acid was used for un­ at —20 °C. Cell fresh weight was determined after specific O-methyltransferases. The standard incuba­ collecting the cells on a fritted glass funnel with suc­ tion mixture consisted of 10 ^1 substrate (THP, tion. All other cell cultures were from our culture 100 nmol, other substrates 50 nmol in DMSO), 20 [d collection and were grown under identical conditions 14CH3-S-adenosylmethionine (0.1 ^iCi/102 nmol, as given above. Amersham), appropriate amount of enzyme and potassium phosphate buffer (0.1 to 0.125 m , pH 7.5) Chemicals to give a final volume of 100 [a1. For monitoring the (/?,5)-Reticuline, (/?,5)-laudanine, (/?,S)-orien- activity of enzyme from column fractions, SAM at a taline perchlorate, (/?,S)-nororientaline-HCl, ( R,S)- concentration of 26 nmol/20 [0,1 per assay (0.05 |oCi) noriso-orientaline-HCl, and (/?,S)-norprotosino- was used. Incubation was carried out for 40 min at 30 °C. The reaction was stopped by addition of 400 jxl 1 m KCl in 1 m phosphate buffer (pH 8) at 4 °C and 500 ^,1 ethyl acetate. The mixture was ro­ tated for 15 min at room temperature and centrif­ uged (Eppendorf system). An aliquot (300 (a.1) of the ethyl acetate was removed and the radioactivity determined in 5 ml Bray’s solution. For caffeic acid, the pH of the incubation mixture was 6.8 and the

reaction stopped by the addition of 400 (il 2 n HC1. R. r 2 r 3 r 4 r 5 An alternative assay, for NMT activity only, con­ Norlaudanosoline H HHH H sisted of 15 (xmol potassium phosphate buffer Laudanosoline HH H H c h 3 (pH 7.5), 38 nmol (/?,5)-norreticuline-HCl in Norreticuline c h 3 HH c h 3 H Reticuline c h 3 HH c h 3 c h 3 DMSO (final conc. 5% v/v), 15 nmol Na ascorbate, Nororientaline c h 3 H c h 3 HH 10.4 nmol 14CH3-SAM (2.4 ^Ci/|xmol) and up to c h H c h 3 H c h 3 Orientaline 3 100 |xg enzyme in a total volume of 150 jaI. Incuba­ Norprotosinomenine H c h 3 H c h 3 H tion was for 60 min at 30 °C. The incubation was Protosinomenine H c h 3 H c h 3 c h 3 Norisoorientaline H c h 3 c h 3 HH terminated by the addition of 200 fil 0.2 m Na2C 0 3/ Isoorientaline H c h 3 c h 3 H c h 3 N aH C 03, pH 10, and 500 ^1 isoamyl alcohol. The Tetrahydropapaverine c h 3 c h 3 c h 3 c h 3 H mixture was rotated for 20 min at room temperature. Laudanosine c h 3 c h 3 c h 3 c h 3 c h 3 After centrifugation, 300 ^il of the organic phase was 1H = a (5); 1H = ß = (/?). taken and the radioactivity determined. 128 Chi-Kit Wat et al. • Norreticuline N-Methyltransferases

Efficiency of extraction of laudanosine evaporation of the solvent and excess Me I, the re­ Two methods were used to determine the efficien­ sidue was recrystallized first from methanol-Et20 cy of EtOAc extraction of laudanosine, the N-methy- (873 dpm/fxmol) and then from methanol (836 dpm/ lated product of THP, from the reaction mixture as Umol), m.p. 215-216 °C, U V ^ H = 280 nm (4.75). assayed above. The first method involved a large MS (80 eV, direct inlet) mle (%): 372(M^,2), scale incubation consisting of 14CH 3-SAM, the en­ 207(15), 206(100), 191(12), 190(18), 151(8), 142(22), zyme and THP. After purifying the labelled product 127(20), 58(72), corresponding to the N-methyl by TLC with solvent systems I and II (see below) it laudanosine ion of laudanosine methiodide. was dissolved in DMSO. An aliquot (10 \x\) of this labelled compound was added to vials containing Enzyme purification varied amounts of unlabelled laudanosine All buffers used were at pH 7.5 containing 20 mM (0 —200 nmol) in the extraction buffer and the solu­ ß-mercaptoethanol unless otherwise stated. Typical­ tion was mixed with EtOAc as described above. The efficiency of the extraction was found to be over 95% ly, 300 g of frozen cells were stirred for 30 min in when the concentration of laudanosine present was 450 ml 0.1 M potassium phosphate buffer containing 60 mM ß-mercaptoethanol. After filtering through up to 25 nmol. In the second method, 20 pil of assay cheesecloth, the filtrate was centrifuged at 27,000 x g mixture was applied on a Sil G/UV 254 Polygram plate for 10 min. The supernatant was fractionated with (Macherey and Nagel, Düren) and developed in sol­ (NH 4)2S 0 4 and the protein which precipitated be­ vent system II. The chromatogram was then scanned tween 35—70% saturation was dissolved in 40 ml of (Berthold, Wildbad). The ratio of activity of the re­ the extraction buffer. The solution was stirred for sidual SAM which remained at the origin to that of 10 min with 7 g Dowex 1x4 (100—200 mesh, phos­ [14C]laudanosine was calculated by cutting out and phate form) and filtered through sintered glass fun­ weighing the area under the peaks obtained from the nel. Dextran-coated charcoal (200 mg) was added to scan. The amount of activity in laudanosine obtained the filtrate, stirred for 10 min and centrifuged. The by this TLC method was found to correspond within supernatant was dialyzed against 0.05 m phosphate ± 20% to that determined by the EtOAc extraction method. buffer. The dialyzed enzyme was passed through a DEAE-cellulose column (2.5 x 15 cm, Whatman DE-52). After washing with starting buffer, a linear Identification of methylated products gradient (0—0.5 m KC1) in 0.05 m phosphate buffer For the identification of benzylisoquinolines, MN- was applied. Fractions of 5 ml were collected at a Polygram Sil G/UV 254 was used with the following flow rate of 1 ml/min. NMT and OMT activities in solvent systems: (I) CHC13: methanol 20:1, (II) to­ the fractions were monitored by assays with (R,S)- luene: EtOAc: diethylamine 7:2:1, (III) CHC1 3 a c e ­ THP, (/?,S)-NRK, and caffeic acid. The fractions tone: diethylamine 5:4:1, (IV) cyclohexane: diethyl­ containing NMT activity which were eluted with the amine 9:1, (V) xylene: methylethyl ketone: metha­ starting buffer were pooled (NMT-I) and dialyzed nol: diethylamine 20:20:3:1, (VI) acetone:metha­ against 0.02 m phosphate buffer. nol: HCOOH: H20 5:3:1:1, (VII) EtOAc: methyl­ The active fractions of NMT-I were subsequently ethyl ketone:HCOOH:methanol 12:12:3:2, (VIII) passed through a column of CM-cellulose (1x8 cm) EtOAc: methylethyl ketone: HCOOH: H20 6:2:1:1. equilibrated with dialysis buffer. The N-14CH3 product of THP (laudanosine) was NMT-II was eluted from the DEAE-column at further identified by co-crystallization with about 0.12 m KC1; details for this enzyme will be laudanosine. The labelled product obtained by incu­ published later. bation with 14CH3-SAM was first purified by TLC with solvent systems II and I. Laudanosine (52 mg) was added, and the mixture recrystallized from Molecular weight determination EtOH (919 dpm/nmol), hexane (823 dpm/|amol) and The NMT-I enzyme, after it was eluted from its petrol (830 dpm/fxmol). This labelled laudanosine respective cellulose columns, was dialyzed against was further converted to laudanosine methiodide ac­ 0.1 m phosphate buffer. The enzyme (1.8 ml) to­ cording to the procedure described in [12]. After gether with ferritin (0.5 mg, 450 kD), cytochrome c Chi-Kit Wat et al. ■ Norreticuline N-Methyltransferases 129

(2 mg, 12.5 kD), and glycerol (0.2 ml) was applied (/?,5)-THP, and finally, with 30 ml starting buffer to an Ultrogel AcA-44 column (2.3 x 88 cm) equili­ containing 50 jam SAM, 10 [am (/?,S)-THP and 0.1 m brated with the dialysis buffer. Fractions of 3 ml KC1. Fractions of 3 ml were collected at a flow rate were collected at a flow rate of 24 ml/h. The column of 0.3 ml/min. was previously calibrated with blue dextran, catalase (240 kD ), aldolase (158 kD), bovine serum albumin (68 kD) or albumin (43 kD), chymotrypsinogen A NMT for preparative purpose (25 kD) and cytochrome c (12.5 kD) (Boehringer, Frozen tissue of B. wilsoniae var. subcaulialata Mannheim). The markers were measured either by (100 g fresh weight) was suspended in 250 ml of their light absorption, catalytic activity, or both. 0.05 m phosphate buffer, pH 7.4, containing 20 mM ß-mercaptoethanol and stirred at room temperature to homogeneity. The mixture was pressed through Affinity chromatography cheesecloth and the filtrate centrifuged for 10 min at The enzyme preparation (NMT-I) used in this 18,000 x g. (NH4)2S04 was added to the extract un­ chromatographic step had been prepurified by two til 70% saturation was reached. After centrifugation subsequent DEAE-cellulose columns and was dialyz- the pellet was resuspended in 14 ml starting buffer ed against 0 .0 2 m phosphate buffer, the starting buf­ and desalted on a Sephadex G-25 column fer for all columns used below. The protein concen­ (2.3 x 18 cm) equilibrated with 0.01 m phosphate tration of this preparation was 0.6 mg/ml. (i?)-THP buffer, pH 7.4. The protein-containing fraction was was used as the substrate in the assays. subjected to ion exchange chromatography (DEAE- SAH-AH-Sepharose 4B: The ligand SAH was Sephacel, Pharmacia; 1.6 x 12.5 cm) and eluted coupled to AH-Sepharose 4B by the procedure de­ with a KC1 gradient (0—400 m M ) in 0.01 m phosphate scribed in [13] and the residual amino groups on the buffer. Fractions between 0.14 and 0.18 m KC1 were gel were blocked by a further carbodiimide reaction pooled and used as enzyme source for the prepara­ with acetic acid. After applying the enzyme solution tive synthesis of labelled N-methylated products; this (3.2 ml), the column (0.9 x 5 cm) was first washed preparation was devoid of any O-methyltransferase with 16 ml 0.02 m KC1 in starting buffer containing activity using caffeic acid or laudanosoline as sub­ varied amounts of SAM followed by 16 ml 0.5 m KC1 strate. In this case, (7?,S)-NRK was used as the sub­ in starting buffer. Fractions of 1.5 ml were collected strate and the alternative enzyme assay given above at a flow rate of 0.3 ml/min. was employed. For protein determinations the mod­ SAH-CH-Sepharose 4B. The affinity gel was pre­ ified Folin method [15] was used with bovine serum pared as described in [14] and equilibrated in the albumin as reference. starting buffer. Column dimensions and enzyme ap­ plication/elution were as described above. (i?,5)-THP-CH-Sepharose 4B. The preparation of Results this matrix was essentially the same as described in [14] with (/?,S)-THP substituting for SAH. The UV General survey of cell cultures for NMT spectrum of the buffer was scanned between Using ( R )- and (S)-THP as substrates, a survey for 200—350 nm at the beginning and at the end of the N-methylating activity was made on more than 50 reaction time. There was a 40% decrease in the ab­ cell suspension cultures propagated from plants that sorbance of the peak at 230 nm. This was taken as an produce isoquinoline alkaloids. Under the conditions indication that (i?,S)-THP was removed from the sol­ used in the assay, the preparations from the genus ution and bound to the gel. The column Berberis showed the highest activity towards these (0.9 x 8 cm) was equilibrated with the starting buf­ two substrates, and consistently with higher activity fer containing 50 [am SAM. The enzyme solution toward the (/?)-isomer (Table I). Cultures from (5 ml), to which SAM was added to give a final con­ other genera such as Corydalis, Eschscholtzia, centration of 50 (am, was then applied. The column Fumaria, and Thalictrum gave preparations with less was successively washed with 30 ml starting buffer activity. As controls, preparations from tobacco and containing 50 (am SAM, 30 ml starting buffer, 10 ml carrot cell suspension cultures were used and neither starting buffer containing 40 SAM and 10 (am one possessed NMT activity towards THP. 130 Chi-Kit Wat et al. ■ Norreticuline N-Methyltransferases

Table I. A survey of (/?)- and (S)-tetrahydropapaverine says were carried out with the EtOAc extraction specific N-methyltransferase activity from Berberis cell cul­ method. Though a 84-99% recovery of laudanosine tures, grown in Linsmaier and Skoog medium [9], in the concentration range from 0—200 nmol was ob­ Cell cultures Days of Specific (pkat/mg tained, however, the extraction of only partially growth activity protein) methylated products with hydroxyl groups still pre­ (R) THP (S) THP sent was not complete by this procedure.

B. Coryi 6 1.7 0.5 By DEAE-cellulose chromatography with 0.5 m B. henryana 16 5.0 2.4 phosphate as starting buffer, two pools of enzymes B. heteropoda 16 7.3 2 . 1 with N-methylation activities were obtained (Fig. 1). B. stolomfera 7 1 2 . 0 7.7 B. vernae 1 1 3.8 2 . 8 The first one was eluted with the starting buffer. On B. vulgaris 1 1 5.1 2 . 2 pooling the most active fractions from this peak, this B. wilsoniae var. sub. 8 29.3 13.0 enzyme (NMT-I) showed activity towards ( R )- and controls: (S)-THP, (fi,S)-NRK, and (R,S )-NLS only. The sec­ Nicotiana tabacum 13 0 0 Daucus carota 7 0 0 ond peak was eluted from the column with a KC1 gradient at about 0.12 m KC1. This protein solution containing NMT-II possessed activities for N- and O- methylation. In this paper only the purification and Purification of NMT from B. vulgaris characterization of NMT-I will be given. The enzyme was first prepurified by (NH4)2S04 Subsequently, an attempt was made to further precipitation, Dowex-A and dextran-coated charcoal purify NMT-I by affinity chromatography. Three af­ treatment. The enzyme preparation thus obtained finity matrices were prepared: SAH-AH-Sepharose showed methylation activities towards (/?)- and (5)- 4B, SAH-CH-Sepharose 4B and THP-CH-Sepha- THP (N-methylation), (/?,S)-norlaudanosoline, rose 4B. With SAH-CH-Sepharose, the enzyme was (/?,S)-norreticuline (O- and N-methylation), ( R,S )- not retained. With THP-CH-Sepharose all the pro­ laudanosoline and caffeic acid (O-methylation). As­ tein was retained but the enzyme could not be

9

-8

■7

-6

-5 1

Fig. 1. Elution profile of NMT-I and NMT-II as well as unspecific O-methyltransferase activities from B. vulgaris cell cultures on DEAE-cellulose columns with (i?,5)-tetrahydropapaverine as substrate (standard assay condi­ tions). Chi-Kit Wat et al. • Norreticuline N-Methyltransferases 131

Table II. Purification procedure for N-methyltransferase-I from B. vulgaris cell suspension cultures [(/?)-THP as sub­ strate].

Purification step Total activity Total protein Specific activity Recovery Purification [pkat] [mg] [pkat/mg] [%] - fold

Crude dialysate 114 572 0 . 2 1 0 0 1 Ammoniumsulfate-precipitation (35-70% ) 140 127.4 1 . 1 123 5.5 DEAE-Cellulose chromatography 43 27.0 1 . 6 38 8 CM-Cellulose chromatography 38 1 0 . 6 3.6 33 18 SAH-AH-Sepharose chromatography 5.5 0.27 20.4 5 1 0 2

eluted, neither when SAM was omitted from the buf­ filtration to have a molecular weight of about 68,000. fer nor when SAM (50 [am ), (i?,5)-THP (10 [am ) and Under standard assay conditions the time course of KC1 (1.0 m ) were present. With SAH-AH-Sepha- the reaction of the enzyme was linear at least up to rose, the NMT was retained on the column but could 1 hr. For the NMT-I enzyme there is a sharp pH not be eluted by the inclusion of SAM in the buffer optimum at 7.65 (Fig. 2). The purified enzyme shows (up to 0.1 m M ). It was, however, released by a KC1 a temperature optimum at 35 °C. KM for (/?)-THP as gradient or by first washing with 0.02 m KC1 followed substrate was in the range of 0.2 m M , while the KM by 0.5 m KC1 in starting buffer. The recovery of the for SAM was 0.04 mM . The concentrations of SAH activity was only 5% with about 5-fold purification compared with the previous step and 102-fold over­ all. A typical purification procedure is summarized in Table II. NMT-I was stable for at least two weeks when kept at 4 °C; on freezing the catalytic activity was, however, totally lost. Attempts to stabilize the enzyme with varying amounts of sucrose, glycerol or DMSO at -2 0 °C failed.

Properties o f N M T-1 The ca. 100-fold purified sample of the NMT-I en­ zyme was used to determine its catalytic properties as shown in Table III. The enzyme was found by gel

Table III. Some properties determined for purified NMT-I from B. vulgaris cell suspension cultures.

Molecular weight 6 8 kD pH-optimum 7.6

Temperature optimum 35 0

K m for (/?)-THP* 180 jam

K m for (S)-THP* 30 [AM PH K m for SAM** 40 [a m Fig. 2. pH profile of the catalytic activity of purified N- SAH concentration for 50% inhibition 80 [AM methyltransferase I from B. vulgaris cell cultures; A — A citric acid/Na-acetate; • — • KH 2PO 4/K 2HPO 4; 0 — 0 * at 1 m M SAM. Tris/HCl at 0.05 m . Standard assay conditions with ( R )- ** at 1 mM of (/?)-THP. THP as substrate. 132 Chi-Kit Wat et al. ■ Norreticuline N-Methyltransferases

Table IV. Substrate specificity of 100 fold purified NMT-I (/?)-THP. As given in the data of the methods sec­ from B. vulgaris cell cultures. tion, the product was purified to a constant specific Substrate Relative Reaction activity and converted to a crystalline derivative activity* product (methiodide) which carried the same specific radio­ [%] activity as the reaction product. The identity of the other reaction products were determined by co­ (/?,5)-Norlaudanosoline 16 laudanosoline (R, S)-Norreticuline 190 reticuline chromatography using several TLC systems. (R, S)-Nororientaline 190 orientaline Several isoquinoline alkaloids regarded as inter­ (R, S)-Noriso-orientaline 3 nd mediates or end products were tested with respect to (/?,S)-Norprotosinomenine 0 - their influence on the methylation reaction catalyzed (/?)-Tetrahydropapaverine 1 0 0 laudanosine (S)-Tetrahydropapaverine 2 0 laudanosine by NMT-I with (/?)-THP as substrate. At 1 m M con­ Norboldine 0 - centration, only (/?,S)-laudanine and berberine (a Norglaucine 0 - metabolite produced by the Berberis cells) inhibited Caaverine 0 — Tyramine 0 - the reaction significantly (by ca. 60%). Neither dop­ Dopamine 0 - amine, 3,4-dihydroxyphenylacetaldehyde, (R,S),- 3-Methoxy-4-hydroxytyramine 0 - norlaudanosoline, (/?,S)-laudanosoline, (R,S)-re- 3,4-Dimethoxytyramine 0 - ticuline, (/?,S)-orientaline, glaucine nor boldine Caffeic acid 0 — showed any drastic inhibition. Furthermore, the * Relative to (/?)-THP; nd = not determined. reaction was not significantly inhibited by usual in­ hibitors of enzyme reactions (at 1 mM conc.), such as: iodoacetamide, iodoacetic acid, N-ethylmale- required to give 50% inhibition of the reaction with imide, 5,5'-dithiobis (2-nitrobenzoic acid), o-iodoso- (/?)-THP as substrate was 80 for NMT-I. Sub­ benzoic acid, p-hydroxymercuribenzoate, KCN, strate specificity for NMt-I is depicted in Table IV. NaN3, sodium diethyldithiocarbamate, a,a,-dipyri- The best substrates so far tested were norreticuline dyl and EDTA. and nororientaline followed by the standard sub­ strate used here, (7?)-THP. (S)-THP was methylated NMT for preparative purposes from B. wilsoniae at about the same rate as norlaudanosoline. Rigor­ ous proof of the reaction product was made for The richest source for NMT was found to be a laudanosoline formed by enzymatic methylation of subspecies of B. wilsoniae (Table I). From this mate­

Fig. 3. Elution profile of N- methyltransferase from B. wil­ soniae var. subcaulialata on DEAE Sephacel [(/?,5)-NRK as substrate] (standard assay conditions). Chi-Kit Wat et al. ■ Norreticuline N-Methyltransferases 133 rial the enzyme was isolated in 37% yield and with a fluorescent properties and chromatographic mobility six-fold purification. [8]. A similar incubation with the non-penolic ( R,S )- Ammonium sulfate precipitation (0—70%), desalt­ THP provided labelled laudanosine [8]. These exper­ ing with Sephadex G-25 and DEAE ion exchange iments indicated that under cell-free conditions O- chromatography yielded the pool fraction (Fig. 3) and N-methylation reactions occur at the expense of with 16.6 mg protein and a specific activity of SAM. From our previous work [3, 6] it is now clear 173 pkat/mg protein. At this stage the enzyme was that several distinct O-methyltransferases in benzyl­ already free of any O-methyltransferase. The ion ex­ isoquinoline synthesis exist in plant tissues. Studies change chromatography step (Fig. 3) was found to of their characteristics have shown that these en­ be absolutely necessary. Prior to this step, the en­ zymes possess a much narrower substrate specificity zyme is strongly inhibited by impurities in the protein than previously imagined. In order to understand ful­ preparation and yields of N-methylated product do ly the enzymatic formation of reticuline, we have not exceed 20%. Using this enzyme preparation isolated and characterized the norreticuline-N- (550 pkat), quantitative conversion of 125 [a,Ci methyltransferase from B. vulgaris. THP and NRK (2.1 nmol) [14CH3]-SAM, and in a separate experi­ was used as substrate for the assay of this enzyme. ment, of 5 mCi (57 nmol) [CJH3]-SAM in the pres­ THP was used for a general survey of different plant ence of at least a 10-fold excess of unlabelled (/?)- or cell cultures in order to exclusively detect only N- (S)-norreticuline as a cosubstrate in a total volume of methyltransfer reactions and to exclude any interfer­ 28 ml containing each 5 mM ascorbate and phosphate ence by O-methylation reactions. Later on with the buffer pH 7.4 was observed. Reticuline was recov­ B. wilsoniae tissue, however, no O-methyltrans­ ered from the incubation mixture by ethylacetate ex­ ferase activity with NRK as substrate was detected traction (at pH 8.5) and was the sole reaction prod­ and thus NRK was used as substrate for the N- uct as judged by TLC and derivative (acetate) forma­ methyltransfer reaction in an alternative assay. A tion. The final specific activities of the products ob­ survey for the enzyme led to the detection of N- served were determined by the “self-displacement” methyltransferase activity in several cell cultures of method using a radioimmunoassay specific for ( R )- the genera Corydalis, Eschscholtzia, Fumaria, and (^-reticuline (U. Wieczorek and M. H. Zenk, Papaver, and Thalictrum (to be published), as well as unpublished results) to be about 5 Ci/mmol for [N- in Berberis, where the highest enzyme activity was C3H3]-reticuline and 45 mCi/mmol for [N-14CH3]- detected. The enzyme involved was purified 100-fold reticuline. Substrate specificity and other charac- from B. vulgaris and named systematically: S- teristica (pH, and temperature optimum, molecular Adenosyl-L-methionine: (/?,S)-norreticuline N- weight etc.) of this enzyme is almost identical to methyltransferase. Two distinct forms of the enzyme NMT-I from B. vulgaris. were found and named NMT-I and NMT-II. Only NMT-I was further characterized in this paper. Comparison with other types of N-methyltransfer- Discussion ase from other plant sources [18] shows that the en­ Isoquinoline alkaloids comprise the largest group zyme described here has a somewhat lower pH op­ of alkaloids in the plant kingdom [1]. Reticuline, the timum (7.65) than the majority of the enzymes re­ central precursor of most isoquinoline alkaloids, is ported up to now. The activity was not affected by formed by condensation of dopamine with 3,4-dihy- Mg 2+ or EDTA and was not inhibited by SH-inter- droxyphenylacetaldehyde [2] with subsequent O- fering reagents (at 1 m M ). Only tetrahydrobenzyliso­ and N-methylations. In vivo experiments have clear­ quinoline alkaloids served as substrates and none of ly shown that both types of methyl groups are deriv­ the precursor molecules such as dopamine or ty- ed from L-methionine [1]. S-Adenosyl-L-methionine ramine was methylated, thus clearly showing a com­ is presumed to be the immediate methyl donor in plete difference to the previously described tyramine these transmethylation reactions. This concept has N-methyltransferases [19]. The fact that nor-apor- been strengthened by the recent finding that ( R,S )- phine alkaloids were not N-methylated by the enzy­ norlaudanosoline in the presence of 14CH3-SAM and mes demonstrates their unique involvement in N- cell-free extracts derived from Berberis aggregata cal­ methyl-benzylisoquinoline alkaloid biosynthesis. lus lead to a compound similar to palmatine in its These enzymes are most active with norreticuline 134 Chi-Kit Wat et al. ■ Norreticuline N-Methyltransferases and nororientaline as substrates. Both stereoisomers for the chemical N-methylation of norbenzyliso- of THP as well as NRK are methylated. This rem ark­ quinolines [17]. However, these are cumbersome if able lack of substrate stereospecificity of these enzy­ material highly labelled with isotopes of carbon and mes also demonstrates that stereochemical control hydrogen is wanted. This enzyme, NRK N-methyl- during the biosynthesis is neither exerted at the first transferase, just described from B. wilsoniae, can be O-methylation [18] nor at the N-methylation level. obtained easily in satisfactory yield (Fig. 3) and it has The much lower activity (ca. 10%) shown towards no absolute preference for either the ( R )- or (S)- (/?,S)-norlaudanosoline indicates that some level of norcompound. Starting with (5)- or (/?)-NRK, the N- O-methylation might be necessary before N-methy­ methyl group of stereochemically pure reticuline lation could take place in vivo. Both observations are could be labelled with 2H,3H,13C or 14C. in absolute agreement with results obtained by The degree of isotope labelling obtained would be Brochmann-Hanssen et al. [5]. By in vivo feeding of the same order as that of commercially available experiments using opium poppy plants, they demon­ methyl-labelled-SAM or that obtained enzymatically strated a low order of stereoselectivity and that O- from labelled L-methionine. This procedure will methylation precedes N-methylation. The lack of N- prove to be superior for obtaining labelled reticulines methylating activity for norprotosinomenine and for biosynthetic purposes to chemial methods [17] norisoorientaline by this enzyme further demon­ used hitherto to resolve enantiomers of this impor­ strates that a 7-O-methyl substitution pattern renders tant intermediate. the benzylisoquinoline molecule almost inactive for N-methylation. Methylation at the 4' OH group (NRK) or at the 5' OH group (nororientaline) has A cknowledgements absolutely no effect on this SAM-dependent trans- Our thanks are due to Profs. Brochmann-Hans­ methylase. sen, Cave, and Nagakura for providing rare al­ For biosynthetic studies there is a definite need to kaloids. Financial support by SFB 145 of Deutsche have available benzylisoquinoline precursors carry­ Forschungsgemeinschaft, Bonn, as well as literature ing a label in the N-methyl group such as reticuline or provision by Fonds der Chemischen Industrie is related compounds [16]. There are several methods gratefully acknowledged.

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