Plant Physiol. (1992) 100, 826-835 0032-0889/92/100/0826/10/$01.

00/0

Received for publication April 1, 1992 Accepted May 15, 1992

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus1

n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo
Naruhiro Hibi, Toshihiro Fujita2, Mika Hatano3, Takashi Hashimoto*, and Yasuyuki Yamada
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan
ABSTRACT

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the Km values for putrescine and S-adenosyl-L-methionine are 277 and 203 Mm, respectively, and the K; value for S-adenosylL-homocysteine is 110 Mm. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2aminonorbornane were most inhibitory, with respective K; values of 11.0, 9.1, and 10.0 Mm. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mm of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6,6-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.

Particular solanaceous genera, such as Atropa, Datura, Du-

boisia, Hyoscyamus, and Scopolia, are characterized by the presence of the pharmacologically important, anticholinergic alkaloids hyoscyamine and scopolamine. These tropane alkaloids are esters of 3a-hydroxytropane derivatives and
l This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (No. 2660090 to T.H.).

phenylalanine-derived tropic acid. Other hydroxytropanes esterified with organic acids of various chemical structures are also found in the Solanaceae and, sporadically, in other taxonomically unrelated families as well (28). Early studies indicated that biosynthesis of MP4, an established precursor of hyoscyamine, does not proceed by way of putrescine (23). The main evidence was that feeding labeled ornithine to Datura plants resulted in asymmetric labeling of the pyrrolidine ring of hyoscyamine (21, 22, 30). Thus, involvement of symmetric intermediates such as putrescine in the formation of MP from ornithine was considered very unlikely. Other supportive evidence for an asymmetric pathway was the incorporation of b-N-methylornithine into hyoscyamine in Atropa and Datura plants (1, 3) and isolation of radioactive b-N-methylornithine after feeding labeled ornithines to Atropa plants (18). Although some of the above evidence was questioned (see ref. 17 for detailed discussion), it had generally been accepted that ornithine is first methylated at the bN position, then decarboxylated to MP, which is further converted to various tropane alkaloids. Recent reinvestigation of this pathway using alkaloid highproducing root cultures of Hyoscyamus albus, however, led to a quite different conclusion. First, the pyrrolidine ring of hyoscyamine was labeled symmetrically after feeding [5-14C]ornithine to the root culture (14). Furthermore, changes in the enzyme activities of arginine decarboxylase, ornithine decarboxylase, and PMT, as well as changes in the contents of tropane alkaloids and diamines during the growth of cultured H. albus roots (16), were consistent with the pathway in which MP is synthesized from ornithine and/or arginine by way of putrescine (Fig. 1). Feeding studies with labeled omithine, arginine, and putrescine, and the inhibitory effects of a-difluoromethylornithine, a potent suicide inhibitor of ornithine decarboxylase, on alkaloid biosynthesis (17) also supported the 'putrescine" pathway. Similar results were also obtained in root cultures of Datura stramonium and Atropa belladonna (36), indicating that this revised pathway may be operating generally in many alkaloid-producing genera. Biosynthesis of nicotine in tobacco is also known to be by way of the putrescine pathway (20).
4Abbreviations: MP, N-methylputrescine; PMT, putrescine Nmethyltransferase; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine.
826

2 Present address: Kishuseishi Co., Ltd., 4-22-1, Minamisuita, Suita 565, Japan. 'Present address: Nihon CIBA GEIGY Co., Ltd., 10-66, Miyukicyo, Takarazuka 665, Japan.

PUTRESCINE N-METHYLTRANSFERASE IN ALKALOID BIOSYNTHESIS

827

11

HOOC

NH2

NHCNH2

HOOC

NH2

L-Arginine

L-Ornithine

3
NH
------

12
nNH2

o Conjugated putrescines
NH2

H8NH2 Agmatine
NH2

Putrescine

Figure 1. Metabolism of putrescine in several alkaloid-producing plants. After being synthesized from arginine and/or ornithine, putrescine is metabolized to polyamines, to conjugated forms, or to alkaloids such as nicotine and tropane derivatives. Some of the enzymes involved are indicated. 1, Arginase; 2, ornithine decarboxylase; 3, arginine decarboxylase; 4, PMT; 5, spermidine synthase.

//5

2N

N2

Spermidine

NH

NH2

N-Methylputrescine

CH3

Spermine

l~CH3
N
0-Tropyl

Tropane alkaloids

Nicotine

Because putrescine is metabolized to the ubiquitous polyamines spermidine and spermine in plants, PMT would function as the first committed enzyme to alkaloid pathway that diverts the metabolism of putrescine from polyamine synthesis to alkaloid synthesis. To evaluate the essential role of PMT on the control of cellular nitrogen flux and to gain further in vivo evidence for the operation of the putrescine pathway, we need to manipulate the in vivo activity of PMT by its specific inhibitor. Administration of such an inhibitor to the root culture was shown here to have profound effects on the metabolism of alkaloids and polyamines.
MATERIALS AND METHODS
Chemicals

containing 75 mL of auxin-free B5 medium (9) supplemented with 3% (w/v) sucrose, then cultured for 7 d. After being harvested with a suction filter, the roots were immediately frozen with liquid nitrogen. The frozen roots were kept at -200C until use. Hairy roots transformed with Agrobacterium rhizogenes (strain 15834) were also cultured in auxin-free B5 medium. All the other roots used have been maintained in our laboratory for several years under conditions described elsewhere (16).

SAM was purchased from Sigma and used after purification as described by Audubert and Vance (2). Putrescine and SAH were also purchased from Sigma. EAH-Sepharose was purchased from Pharmacia, and SAH was coupled to the gel according to the manufacturer's instructions to give SAHSepharose. The other amines were obtained from Sigma, Aldrich, Nacalai tesque (Tokyo), and Wako Pure Chemical Industries (Osaka, Japan). [methyl-3H]SAM (147 kBq/mmol) was purchased from Amersham.
Plant Materials

Buffers The following buffers were used: (A) 100 mm K-phosphate buffer (pH 7.5) containing 0.5% (w/v) sodium ascorbate, 5 mM EDTA, 5 mm DTT, 0.05 mm iodoacetate, 0.05 mm diisopropyl fluorophosphate, and 1 mg/L pepstatin; (B) 50 mm Kphosphate buffer (pH 7.5) containing 1 mm EDTA, 1 mM DTT, 0.01 mm iodoacetate, 0.01 mm diisopropyl fluorophosphate, and 0.1 mg/L pepstatin; (C) 20 mm Tris-HCl buffer (pH 7.5) containing 1 mim EDTA and 1 mm DTT; (D) 50 mM Tris-HCl buffer (pH 7.5) containing 1 mm EDTA, 1 mm DTT, 10% (v/v) ethanol, and 0.1% (w/v) Tween 20.
Purification of PMT All purification procedures were carried out at 40C. Frozen roots of H. albus were homogenized thoroughly with a Polytron (Kinematica, Luzem, Switzerland) in buffer A containing 10% (w/v) insoluble PVP (Polyclar AT; Kasei-hin Kohgyo, Osaka, Japan). The homogenate was passed through a composite cheesecloth-Miracloth (Calbiochem)-cheesecloth filter and centrifuged at 7000g for 30 min. A small portion of the supematant was passed through a PD-10 column (Pharma-

Plants were grown in a growth chamber at 200C and 80% RH with a 14-h light period, and harvested at the flowering stage for enzyme extraction. Roots of Hyoscyamus albus have been maintained in vitro in our laboratory (15). Prior to the experiments reported here, aliquots of these roots were transferred to 300-mL flasks

828

HIBI ET AL.

Plant Physiol. Vol. 100, 1992

cia) using buffer C, this desalted eluate subsequently being referred to as crude extract. Solid KCI was added to the supematant at a final concentration of 3 M and stirred for 30 min. After centrifugation at 7000g for 30 min, the supernatant was applied to a Butyl-Toyopearl 650C column (5 x 16 cm; Tosoh, Tokyo) that had previously been equilibrated with buffer B plus 3 M KCl. The column was washed with 1200 mL of the same buffer, and the enzyme was eluted with buffer B at a flow rate of 2.5 mL/min. Active fractions were pooled and subjected to ion-exchange chromatography on a Q-Sepharose column (2.5 x 16 cm; Pharmacia) that had previously been equilibrated with buffer C. The column was washed with 250 mL of the same buffer and the enzyme was eluted using a linear salt gradient of 0 to 1 M NaCl in the same buffer at a flow rate of 2.1 mL/min. Active fractions were pooled and loaded on a SAH-Sepharose column (1.6 x 10 cm) that had previously been equilibrated with buffer D. The column was washed with 60 mL of the same buffer and the enzyme was eluted using a linear salt gradient of 0 to 0.5 M NaCl in the same buffer at a flow rate of 0.4 mL/min. Active fractions were pooled and stored at -200C. This enzyme solution was passed through a PD-10 column before use, using buffer C. Protein concentration was measured according to the method of Bradford (6).

reactivity, the remaining aqueous phase was reextracted with 1 mL of chloroform to check whether the respective methylated products were efficiently transferred to the organic phase. Kinetic Analysis
The kinetic parameters (SD) were calculated by Wilkinson's statistical analysis method (37).

Alkaloid Analysis

Enzyme Assay
PMT activity was routinely measured by the method originally developed by Feth et al. (8). The reaction mixture of 50 AL contained, at a final concentration, 6 mm putrescine dihydrochloride, 2 mm SAM, 100 mi Tris-HCl buffer (pH 9.0), and the enzyme. After 30 min of incubation of 300C, the reaction was stopped by heating at 1000C for 3 min. The reaction product MP was then dansylated. To the incubation mixture, 200,uL of 65 mm borate-KOH buffer (pH 10.5) was added, followed by addition of 150 ,L of dansylchloride solution (5.4 mg/mL acetonitrile). The mixture was heated for 15 min at 560C in the dark, centrifuged briefly, and then applied directly to the HPLC column. Dansylated amines were separated with a 5-mm LiChrospher 100 RP-18 endocapped column (4 x 250 mm; Merck) at a flow of 1.0 mL/ min at 400C. The isocratic mobile phase was a mixture of an aqueous 2% (w/v) H3PO4 solution adjusted to pH 5.2 with triethylamine and acetonitrile at a ratio of 35:65 (v/v). Dansylated amines were quantified by means of a fluorescence detector (excitation at 365 nm and emission at 510 nm). Substrate specificity was studied by the other assay method, which measured the radioactivity in the reaction product formed from amine substrate and radioactive SAM labeled at the methyl group to be transferred (24). For the reaction (50-,uL scale), the unlabeled SAM component in the above reaction mixture was replaced with 1 mm [methyl-3H]SAM (147 kBq/mmol). After the reaction was stopped by the addition of 20 uL of 10% (w/v) NaOH solution saturated with NaCl, 1 mL of chloroform was added and the mixture 'was vortexed for 1 min. The mixture was briefly centrifuged, after which 0.5 mL of the organic phase was transferred to a counting vial containing 3 mL of a toluene scintillation mixture. When different substrate amines were compared for

Samples (50 mg dry weight) were lyophilized, homogenized, and soaked in 4 mL of 0.1 N H2SO4. The homogenates were sonicated for 20 min and filtered through No. 2 filter paper (Advantec, Tokyo). The filtrates were mixed with 0.4 mL of concentrated NH40H, 1 mL of the mixtures were applied to an Extrelut-1 column (Merck), and alkaloids were eluted with 6 mL of chloroform. For the analysis of hyoscyamine, scopolamine, and 6,B-hydroxyhyoscyamine, the chloroform extract was dried at 300C. The dry residues were dissolved in 20 ,uL of N,O-bis(trimethylsilyl)acetamide and 80 AL of a dioxane solution containing tricosane (625 ,g/mL) and analyzed by GLC (Shimadzu model GC-7A) equipped with a CB31-M30-025 capillary column (Shimadzu) at a column temperature of 2350C. For the analysis of tropinone, tropine, and pseudotropine, 100 IuL of a dioxane solution containing tricosane (625 Ag/mL) was added to the chloroform extract, which was then evaporated at 300C until the volume of the solution was reduced to approximately 100 ,uL. The concentrated solution was directly analyzed by GLC. The column was held at 1200C for 8 min, then the column temperature was increased from 120 to 2500C at a rate of 32C/min.

Polyamine Analysis
Polyamines were determined according to the method of Tiburico et al. (34). Samples (37.5 mg dry weight) were homogenized and soaked in 1 mL of 5% (v/v) cold perchloric acid. The homogenates were kept at 40C for 2 h, then centrifuged at 15,000g for 20 min. The supematant was set aside and the pellet was resuspended in 1 mL of 1 N NaOH. The pellet suspension and the original supematant (200 ,uL each) were separately mixed with 200 ALL of 12 N HCl and hydrolyzed for 18 h at 1 10C in sealed vials. The hydrolysates were filtered through glass wool, dried at 700C, then resuspended in 200,uL of 5% (v/v) perchloric acid. The nonhydrolyzed perchloric acid supernatant contained free polyamines, whereas the hydrolyzed perchloric acid supematant and hydrolyzed pellet contained polyamines liberated from conjugates. These fractions were dansylated (31) and analyzed by HPLC on a LiChrosorb RB-18 column (5-,um particle size, 4mm diameter, 125-mm length; Merck) with a programmed solvent gradient of 2% phosphate-triethylamine buffer (pH 5.2):acetonitrile (v/v) from 60 to 90% in 30 min at a flow rate of 0.95 mL/min at 400C. Detection was by means of the excitation at 365 nm and emission at 510 nm. The amounts of conjugated polyamines were calculated by subtracting the polyamines in the supernatant fraction from those in the

PUTRESCINE N-METHYLTRANSFERASE IN ALKALOID BIOSYNTHESIS

H. niger D. innoxia
A. belladonna

829

Distribution of PMT among Various Cultured Roots

Leaf
Stem

Flower
Main root

Branch root
Cultured root
not tested

Cultured cell
0
10 20
30 0

not tested

. ....... ....
.....

10

20

30 0

10

20

30

PMT activity (pkatmg protein)

Figure 2. Distribution of PMT activity in plant

organs.

hydrolyzed perchloric acid supernatant fraction plus hydrolyzed pellet fraction.

n-Butylamine Feeding

Roots (0.4 g fresh weight) of H. albus and D. stramonium cultured on a gyratory shaker at 90 rpm and 250C in the dark in 100-mL flasks containing 25 mL of liquid B5 medium with varying concentrations of n-butylamine (filtersterilized). Roots were harvested after 1 week, then analyzed for alkaloids and polyamines.
were

RESULTS

Distribution of PMT among Plant Organs

PMT activity was measured in several plant organs, cultured roots, and cultured cells of Hyoscyamus niger, Datura innoxia, and A. belladonna (Fig. 2). The activity was found only in the root, with no activity detected in the leaf, stem, flower, or cultured cells. In all species, branch roots showed higher activity than main roots.
.3

Restricted expression of PMT in the root tissues led us to investigate cultured roots of various species for possible correlation between PMT activity and accumulation of alkaloids. Tropane esters of tropic acid were analyzed in this study, and root cultures tested included 17 species representing 6 genera (Hyoscyamus, Physochlaina, Atropa, Physalis, Datura, and Duboisia) from Solanaceae and 1 species (Calystegia sepium) from Convolvulaceae. PMT activity was found in all cultured roots investigated, but the level of the activity varied substantially among species (Fig. 3). When cultured roots that contained hyoscyaminetype alkaloids were compared, a rough correlation was found between PMT activity and total alkaloid content in the root cultures. The correlation coefficient was 0.745 (n = 12, P < 0.01). One exception was Datura roots, in which relatively low PMT activity was detected despite high alkaloid content (mainly hyoscyamine). The genera Physalis and Calystegia did not contain tropane esters of tropic acid but have been shown to produce tropane esters of other acids (Physalis [4, 5]) or polyhydroxylated tropanes (Calystegia [10]). The presence of PMT activity in these root cultures is consistent with their proposed ability to synthesize such tropane derivatives. Detection of PMT activities in all root cultures listed here does not imply that PMT is ubiquitously present in root culture, because we and others could not detect PMT activity in the root cultures of Brassica compestris (16), Raphanus sativus (16), Browallia americana (data not shown), or tomato (25), none of which produce MP-derived alkaloids. For the following studies, cultured roots of H. albus were primarily used because of their high enzyme activity and good growth.
Purification of PMT

The enzyme was partially purified by hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on Q-Sepharose, and affinity chromatography on
Figure 3. PMT activity and the contents of hyoscyamine-derived alkaloids among various root cultures. *, Hyoscyamine; E, 6fl-hydroxyhyoscyamine; scopolamine.
E,

Hyoscyamua albums H. niger H. muticus H. pusillus

H. canariensis H. bohemicus H. turcomanicus Physochialna orientalls Atropa acuminate A. belladonna Physalls edulls P. florldlana P. franchettll P. peruvlana P. purulnosa Datura stramonium Dubolsia lelchhardtfi

I I U
I
~l
0
0.5
.
.
.
0

Calystegila seplum

1.0

PMT activity (pkatmg dry weight)

10 20 30 Alkaloid content

40

(nmol/mg dry weight)

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HIBI ET AL.

Plant Physiol. Vol. 100, 1992

Table I. Purification of PMT from Cultured H. albus Roots

Purification Step
Protein

Total Activity

Specific Activity

Purification

mg
Crude extract 3 M KCI Butyl-Toyopearl Q-Sepharose SAH-affinity
4920 3350 536 272 83

nkat
143 144 51 43 32

pkat/mg
29 43 95 160 385

-fold
1.0 1.5 3.3 5.5 13.3

Yield % 100 101 36 30 22

SAH-Sepharose. This resulted in a 13.3-fold increase in specific activity of the enzyme preparation, with a 22.4% recovery, as compared with the crude extract (Table I). The specific activity of our partially purified enzyme preparation (385 pkat/mg protein) was somewhat lower than the value obtained for partially purified tobacco PMT (850 pkat/mg). In our hands, PMT from cultured H. albus roots was very difficult to purify primarily because the enzyme was always eluted as a broad peak from ion-exchange columns and hydrophobic columns. The reason for this abnormal behavior is not clear. There may exist a microheterogeneity (possibly caused by posttranslational modifications, partial proteolytic degradation, or the presence of multiple genes) in the PMT preparation used in our experiment. Glycosylation is one common form of posttranslational modification, although H. albus PMT did not bind to the Con A-affinity column (Pharmacia) and the Glycan Detection Kit (Boehringer Mannheim GmbH) did not detect glycoproteins coeluting with PMT activity during column chromatographies. Inclusion of several proteinase inhibitors in the extraction buffer did not improve the chromatographic behavior of PMT. Furthermore, H. albus PMT was inactivated in the presence of ammonium sulfate. Thus, we used a high concentration (3 M) of KCl, instead of much more common ammonium sulfate, in the buffer for hydrophobic chromatography. PMT from cultured roots of D. stramonium was also sensitive to ammonium sulfate (N. J. Walton, personal communication), but such sensitivity was not reported for tobacco PMT during its partial purification (24).
Mol Wt and pH Optima

intersecting lines were obtained when the SAM concentrations were varied in a series of fixed putrescine concentrations (Fig. 4B). Intercept and slope replots versus reciprocal fixed substrate concentrations generated straight lines (data not shown). Secondary intercept plots were linear and yielded the Km values of 277 10 ,uM for putrescine and 203 20 ,iM for SAM. These initial velocity data indicate a mechanism of sequential binding. The Km values are similar to those
0.3

A
0.2

v-

0.1

0.0
0

10

20

1/[SAM] (1/mM)
0.3

0.2

The mol wt of H. albus PMT as determined by gel filtration on TSK-GEL G3000SW (Tosoh, Tokyo) was 62,100 700 (mean of four measurements SD), which is similar to the mol wt (60,000) reported for PMT from tobacco (24). PMT activity was measured over a pH range of 4.0 to 10.5. Optimum activity was found at pH 9.0 in Tris-HCl buffer and at 9.5 in glycine-NaOH buffer. Tobacco PMT and most other N-methyltransferases also have relatively high pH optima between pH 8 and 9 (27), which may be related to the reaction mechanism of the N-methyltransferases in general (see 'Discussion').

v-

0.1

0.0
0

10

20

1/[Putrescine] (1/mM)
Figure 4. Double-reciprocal plots of initial velocities of PMT reaction. A, Putrescine as a fixed substrate at 1 (0), 0.35 (A), 0.15 (O), 0.1 (0), and 0.05 mm (A) and SAM as a variable substrate. B, SAM as a fixed substrate at 1 (0), 0.25 (A), 0.15 (E), 0.1 (-), and 0.05 mm (A) and putrescine as a variable substrate.

Kinetic Properties

Double-reciprocal plots with varying concentrations of putrescine and several fixed concentrations of SAM gave linear lines that intersect below the abscissa (Fig. 4A). Similar

PUTRESCINE N-METHYLTRANSFERASE IN ALKALOID BIOSYNTHESIS

831

0.5

0.4
-V

0.3
v-

0.2 0.1

0.0
0

10

1/[SAM] (1/mM)

0.4

JC 0T-

0.3 0.2 0.1

0.0
0

at 2 mm (Tables II and III), and the Km values were determined for some substrates. H. albus PMT was most active against amines that have at least two amino groups separated by three or four methylene groups. Of 22 amines tested for methyl transfer, putrescine (1,4-diaminobutane; Table III, No. 18) was the most efficient substrate (Km 277 10 gM), followed by trans-1,4-diaminocyclohexane (No. 25; Km 113 3 8am) and a mixture of cis- and trans-1,4-diaminocyclohexane (No. 24; Km 360 10 Mm). 1,3-Diaminopropane (No. 17) was methylated less efficiently, whereas cadaverine (1,5diaminopentane; No. 19) and 1,6-diaminohexane (No. 20) were inactive. 1,4-Phenylendiamine (No. 26) and 1,4-diaminopiperazine (No. 27) were inactive as well. N-Methyldiamines (N-methyl-1,3-diaminopropane, No. 28; and MP, No. 29) and polyamines (3,3'-iminobispropylamine, No. 21; spermidine, No. 22; and spermine, No. 23) were also methylated to some extent. Monoamines were generally inactive, but slight activities were observed with n-butylamine (Table II, No. 2) and N-methyl-n-butylamine (No. 13). It was reported that when tobacco PMT was assayed for 1,3-diaminopropane, cadaverine, and MP at 4 mm, only MP served as a substrate (8% of the activity with putrescine) (24). Thus, a varying reactivity with 1,3-diaminopropane seems to characterize the PMTs from H. albus and tobacco.

10

20

30

40

1/[Putrescine] (1/mM)
Figure 5. Inhibition of PMT by different fixed concentrations of SAH at varying concentrations of SAM (A) and putrescine (B). The concentrations of SAH used were 2.0 (A), 1.0 (0), 0.5 (O), 0.25 (A), and 0 mM (0).

Table II. Substrate Specificity of PMT and its Inhibition by Monoamines Methylation of various amines was assayed at 2 mm by measuring the product's radioactivity transferred from [methyl-3H]SAM, and expressed as percentage values relative to the methylation of putrescine. Inhibition of the PMT reaction by the same set of amines was assayed by measuring by HPLC the formation of N-methylputrescine from 1 mm putrescine in the presence of amine inhibitors at 1 mm, and expressed as relative values. The values are the means of two replicates. Relative Rates (%) of
No.
I

reported for the enzyme from tobacco roots (24), which had Km values of 400 Mm for putrescine and 110 Mm for SAM, and those reported for the crude enzyme from cultured roots of D. stramonium (8), which had Km values for 880 gM for putrescine and 150 AM for SAM. The order of substrate binding and product release was indicated from product inhibition studies. SAH was found to be a competitive inhibitor with respect to SAM, and a noncompetitive inhibitor with respect to putrescine (Fig. 5), with a Ki value of 110 5 Mm. These kinetic patterns are consistent with an ordered bibi mechanism in which SAM is the first substrate to bind to the enzyme, followed by putrescine, with MP being the first product to be released, followed by SAH. This order of substrate binding and product release is typical of many Nand O-methyltransferases (for example, ref. 19 and references therein).
Substrate Specificity

Structure
-. NH2
%e,

Methylation
0

Inhibition
79

NH2

4
0 0
0

95
63 3
97

3
4

NH2

_,NH2

NH2 NH2

not tested
not tested

2
90

7
8
9

H3C-C NH2

NH2

not tested
not tested

53
9

CN-NH2

10

ATNH2 (endo)

not tested

66

11
12

A--Y9H2
'z-

(exo)

not tested
0
1
0

96
31
20
0

o_ NHCH3 _ NHCH3
NHCH3

13 14
is

ONHCH3
0 NH-

not tested
0

82
7

The specificity for amine substrates were studied by measuring the transfer of the methyl group from SAM to substrates

16

832

HIBI ET AL.

Plant Physiol. Vol. 100, 1992

Table MII. Substrate Specificity of PMT and its Inhibition by Diamines Methylation and inhibition were assayed by the same methods described in Table II.
Relative Rates (%) of
No.
17

Structure
H2N __% NH2

Methylation
16 100 0
0 4

Inhibition
40

18 19
20

H2N-'.'S NH2
H2Ne%_ NH2

not tested

spermidine (No. 22), spermine (No. 23), 1,4-phenylendiamine (No. 26), 1,4-diaminopiperazine (No. 27), and Nmethyl-1,3-diaminopropane (No. 28) showed little or no inhibition. The enzyme activity was inhibited moderately by trans-1,4-diaminocyclohexane (No. 25), whereas a mixture of cis- and trans-1,4-diaminocyclohexane (No. 24) showed
much less inhibition.

(No. 14). At 1 mm, 1,3-diaminopropane (Table III, No. 17) and cadaverine (No. 19) were moderately inhibitory, and 1,6diaminohexane (No. 20), 3,3'-iminobispropylamine (No. 21),

59
12 0 5 7

21
22

H2N'-.~ NH2 H2N-.s._NH--%,NH2

H2N'%._-NH-'N'NNH2
H2N__NH -..%.NH%.-NH2

29

23
24

24

H2N-CNH2(cis,trans)
H2N-CNH2 (trans)

42
74 0

13
78
0
1

Feeding of n-Butylamine to Cultured Roots Strong inhibition of PMT by n-butylamine in vitro prompted us to study its effects on the formation of tropane alkaloids and polyamines in root cultures of H. albus and D.
stramonium.

25
26
27

H2N.&NH2
H2N.NN-NH2

28

H2NA.-NHCH3

25

29

H2NA-%.NHCH3

not tested

Effect of Inhibitors
Several O-methyltransferases require Mg2` for maximum activity (27), but no such requirement for Mg2+ or inhibition by EDTA (10 mnm) was observed for H. albus PMT. Unlike many methyltransferases, sulfhydryl reagents inhibited the activity only at relatively high concentrations: p-chloromercuribenzenesulfonic acid (60% inhibition at 0.1 mM), Nethylmaleimide (32% at 1 mM), and iodoacetamide (24% at 10 mM). At 1 mm, the activity was inhibited slightly (less than 25%) by MgCl2 and CoCl2; moderately (40%) by CaCl2; and strongly (more than 80%) by MnCl2, CuC12, and ZnCl2. Effects of these compounds on tobacco PMT were similar but not the same in that p-chloromercuribenzoate showed 63% inhibition even at 0.01 mm, and Mn2' did not inhibit tobacco PMT at 1 mM (24). Various amines were tested at 1 mm for their inhibitory effects on PMT activity (Tables II and III). The enzyme activity was inhibited strongly by cyclohexylamine (Table II, No. 5), exo-2-aminonorbomane (No. 11), and n-butylamine (No. 2), and moderately by n-propylamine (1) and n-amylamine (No. 3). Kinetic analysis indicated that these monoamines were competitive inhibitors of PMT with respect to putrescine (data not shown), with respective Ki values of 9.1 0.3, 10.4 0.6, 11.0 0.2, 80 10, and 120 20 AM. Inhibition by 4-methylcyclohexylamine (a mixture of cis and trans, No. 7) was strong but less than that by cyclohexylamine. PMT activity was inhibited moderately by trans-2-aminocyclohexanol (No. 8) and endo-2aminonorbomane (No. 12). nHexylamine (No. 4), aniline (No. 6), and dicyclohexylamine (No. 16) showed little or no inhibition. Inhibition by Nmethylmonoamines was weaker than that by corresponding monoamines. The inhibition decreased in the order of Nmethylcyclohexylamine (No. 15), N-methylpropylamine (No. 12), N-methylbutylamine (No. 13), and N-methylhexylamine

n-Butylamine did not severely inhibit the growth of H. albus roots, although some growth inhibition was apparent at increasing concentrations of the monoamine (Fig. 6A). Alkaloids in the root culture were present mostly in the cells. A small proportion (about 2%) of alkaloids detected in the culture medium increased at exogenous n-butylamine concentrations higher than 6 mm. At 10 mm of the inhibitor, about 11% of the alkaloids in the culture were found in the medium. Alkaloids present in the cells and the medium were summed and the total alkaloids in each flask are shown in Figure 6. At 1 mm of the inhibitor, the contents of tropinone, tropine, and pseudotropine drastically decreased by 78, 73, and 69%, respectively, and the hyoscyamine content decreased by 52% at 6 mm, whereas the contents of 6i-hydroxyhyoscyamine and scopolamine remained relatively constant

(Fig. 6A). The n-butylamine feeding also had drastic effects on the polyamine content (Fig. 6, B and C). Polyamines in plants are present in both free and conjugated forms. The conjugated (or bound) polyamines are mostly amides formed with
various organic acids (33). The free polyamines constituted only a small fraction (about 8%) of the total polyamines in the cultured H. albus roots and were, in decreasing order of abundance, composed of MP, putrescine, spermidine, and spermine. In the conjugate fraction, putrescine was highly abundant, amounting to 80% of the conjugated polyamines, whereas the content of MP was very low. The n-butylamine treatment caused a large increase in putrescine content (5fold in the free pool and 2-fold in the conjugated pool), as well as a drastic decrease in the MP content. In both the free and conjugate fractions, the spermine content slightly increased, whereas the spermidine content slightly decreased. The contents of total polyamines (free forms plus conjugated forms) and alkaloids (tropine esters plus alkamine intermediates) are summarized in Figure 6D. The patterns of the increase in putrescine and the decrease in alkaloids were mirror images. The total putrescine pools increased by approximately 40 nmol/mg dry weight, which was large enough to account for the decrease in the total alkaloid pools. To confirm the results obtained in H. albus root culture, nbutylamine was also fed to a root culture of D. stramonium (Fig. 7). Root growth was inhibited by 13% at 1 mm, and by 42% at 3 mm of exogenous n-butylamine. Because of this

PUTRESCINE N-METHYLTRANSFERASE IN ALKALOID BIOSYNTHESIS

833

growth inhibitory effect at higher concentrations, the inhibitor was used at concentrations of 0.5 and 1 mm. Tropane alkaloids in D. stramonium root culture were composed mainly of tropine and hyoscyamine, and a reduction of alkaloid content caused by n-butylamine treatment was evident in the tropine pool, whereas the content of hyoscyamine and MP was not drastically affected by inhibitor concentrations of up to 1 mm. By the same treatment, free and conjugated forms of putrescine significantly increased and those of spermidine slightly decreased. Thus, the addition of 1 mm n-butylamine resulted in a decrease in the combined total content of alkaloids plus MP of 10.9 nmol/mg dry weight, as well as an increase in the combined total contents of putrescine plus polyamines of 11.1 nmol/mg dry weight. This reverse relationship between the contents of putrescine and tropane alkaloids, caused by the inhibitor of PMT, was essentially the same as, although of a lower magnitude than, that observed in the Hyoscyamus root culture, and provided further in vivo evidence that PMT is the first enzyme specific to the alkaloid biosynthetic pathway.
DISCUSSION In this paper, we described detailed properties of PMT from cultured H. albus roots. Our results confirm and substantially extend the report of partially characterized tobacco PMT that functions for nicotine biosynthesis (24). Although relatively minor differences were also noted on their substrate specificity and the effects of some inhibitors, tobacco and H. albus PMTs are basically very similar enzymes, regardless of their roles in the biosynthesis of different alkaloids. In this respect, it is noteworthy that both nicotine and tropane alkaloids are sometimes found in the same species (7) and that recent revision of the early biosynthetic pathway for tropane alkaloids makes the two pathways identical up to the formation of methylpyrrolinium cation. Pathways specific to nicotine and tropane alkaloid biosynthesis might have

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10

20

30

40

50

10

20

30

Content (nmol/mg dry weight)

Figure 7. Effects of n-butylamine on accumulation of alkaloids and polymines in D. stramonium root culture. A, Combined polyamine contents (free plus conjugated forms) in nmol/mg dry weight: ED, putrescine; [, spermidine; *, spermine. B, The contents of MP and alkaloids in nmol/mg dry weight: U, MP; O, tropinone; 3, tropine; U, pseudotropine; *, hyoscyamine. The values are the means of four replicates (SD).

evolved by extension from a common early pathway composed of PMT and a diamine oxidase. PMT activities were detected only in the roots of A. belladonna, D. innoxia, and H. niger. Similar root-specific PMT activities have been reported for tobacco (24). This restricted localization of PMT in the root is consistent with the site of nicotine and tropane alkaloids (35). Recent immunohistochemical studies localized hyoscyamine 6,B-hydroxylase, an enzyme in the late part of tropane alkaloid biosynthetic pathway, to the pericycle of the root with no or little secondary growth (12), which explains the high biosynthetic potential of developmentally young roots, such as branch roots and cultured roots. Future studies will address whether other
80

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120

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80

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2
c

10

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Oa

01.0 0
0

10

10

10

10

n-Butylamine Conc. (mM)

n-Butylamine conc. (mM)

n- Butylamine

Conc. (mM)

Figure 6. Effects of n-butylamine on root growth and accumulation of alkaloids and polyamines in H. albus root culture. The values are the means of three replicates. SDS are shown only in some measurements for clarity. A, Root growth (x) in mg dry weight and alkaloid contents in nmol/mg dry weight: 0, tropinone; A, tropine; O, pseudotropine; *, hyoscyamine; A, 6f-hydroxyhyoscyamine; *, scopolamine. B, Free polyamine contents in nmol/mg dry weight: 0, putrescine; *, MP; A, spermidine; A, spermine. C, Conjugated polyamine contents in nmol/ mg dry weight: 0, putrescine; *, MP; A, spermidine; A, spermine. D, Contents of total alkaloids and combined individual polyamines in nmol/mg dry weight: 0, putrescine; EO, MP; A, spermidine; A, spermine; 0, total alkaloids. Total alkaloids include tropinone, tropine, pseudotropine, hyoscyamine, 6,B-hydroxyhyoscyamine, and scopolamine. Combined polyamines are the sum of free and conjugated forms.

834

HIBI ET AL.

Plant Physiol. Vol. 100, 1992

enzymes of alkaloid biosynthesis (including PMT) are also present specifically at the pericycle. Most of the effects of various amines on PMT as substrates or inhibitors (Tables II and III) can be explained by postulating two substrate-binding subsites and a catalytic subsite in the enzyme's active site (Fig. 8). In this model, putrescine is proposed to bind in a hooked form with one amino group at substrate-binding subsite A and the other amino group at the catalytic subsite C where N-methylation takes place. Subsite A, which accommodates an amino group but not an aminomethyl group, and subsite B, which binds the hydrocarbon unit in putrescine, together contribute mostly to substrate binding. According to this model, monoamines can bind to the active site only in an orientation that places their sole amino group at subsite A, leaving subsite C empty. This mode of binding would make monoamines (especially the ones that occupy subsite B) good competitive inhibitors of putrescine, but poor substrates. The fact that 1,4-diaminocyclohexanes, cyclohexylamine, and the two isomers of 2aminonorbornane bind efficiently to PMT suggests that subsite B is large enough to accept cyclohexane and bornane rings, and is primarily hydrophobic. trans-1,4-Diaminocyclohexane was a more efficient substrate and a stronger inhibitor than a mixture of the cis- and trans-isomers 1,4diaminopiperazine and 1,4-phenylendiamine, suggesting that two amino groups of the substrate diamine are oriented in a trans position when bound to the enzyme. Surprisingly, monoamines were not only capable of binding to PMT, but their affinities to the active site (as judged from their Ki values) were 20- to 40-fold higher than those of corresponding diamines (as judged from their Km values). This indicates that subsite C has a positive charge, provided by the enzyme's amino acid residue(s) or bound SAM, which would dispel an incoming substrate amino group. The relatively high pH optimum for PMT may help to reduce this positive charge and also to convert the amino group of putrescine to a nonprotonated form. The amino group of putrescine is expected to be a nonprotonated form based on the SN2 mechanism proposed for SAM-dependent methyltransferases (29). The inhibitory effects of various monoamines on H. albus PMT reported here are remarkably similar to their effects on spermidine synthase, which catalyzes the transfer of the aminopropyl moiety of decarboxylated SAM to putrescine. For example, the three most potent inhibitors of PMT, cyclohexylamine, exo-2-aminonorbomane, and n-butylamine,

I ( :2

11INNH 2SAM 81 j

Figure 8. Active site model of PMT. A predicted binding mode for putrescine is shown.

were all reported to strongly inhibit spermidine synthase from pig liver (29). A recent detailed study of the various inhibitors and substrates of the synthase has led to a model of its active site (29), which turned out to be very similar to our model on PMT. It should be interesting to compare the primary amino acid sequence of spermidine synthase (26) with that of PMT when it becomes available. The availability of potent inhibitors of H. albus PMT provided us with an opportunity to manipulate the metabolism of putrescine in alkaloid-producing root cultures. Feeding nbutylamine at 1 mm to root cultures caused a large decline in the contents of MP and alkamine intermediates, especially in H. albus. At higher concentrations of the inhibitor, the hyoscyamine content decreased further, but the contents of the metabolites of hyoscyamine (6,B-hydroxyhyoscyamine and scopolamine) remained constant. These changes in metabolite contents indicate that most of the intermediates in alkaloid biosynthesis exist in metabolically active pools and that synthesis of the two hyoscyamine-derivatives is not controlled primarily by the size of the cellular hyoscyamine pool. The activity of hyoscyamine 60-hydroxylase that catalyzes two consecutive reactions from hyoscyamine to scopolamnine (13) may be a major limiting factor for scopolamine synthesis in cultured H. albus roots. The enhanced activity of the hydroxylase in transgenic hairy root cultures of A. belladonna, a typical hyoscyamine-rich species like H. albus, that expressed the hydroxylase transgene of H. niger led to an increased contents of 6f3-hydroxyhyoscyamine and scopolamine (T. Hashimoto, D.J. Yun, Y. Yamada, unpublished results). Most of the increased putrescine resulting from n-butylamine feeding was metabolized to its conjugated forms. Strong putrescine-conjugating activities in cultured H. albus roots had been inferred when 30% of the polyamines derived from labeled putrescine was found in putrescine conjugates after 4 h of label feeding (17). Also noted in the previous feeding study with labeled putrescine (17) was the considerable radioactivity (about 23% of the labeled polyamine metabolites) recovered in spermidine, spermine, and their conjugates. In the present study, however, the inhibitor treatment resulted in an increase in spermine and a decrease in spermidine, but their combined content was not markedly affected despite an increased supply of putrescine. The most plausible explanation would be that n-butylamine also inhibited spermidine synthase in vivo. When protoplasts from Chinese cabbage leaves were treated with cyclohexylamine, spermidine synthesis was inhibited, whereas spermine synthesis was stimulated (11). It has been suggested that cyclohexylamine causes an increase in decarboxylated SAM, which is then utilized for the conversion of spermidine to spermine. The structural similarity between cyclohexylamine and n-butylamine indicates that both monoamines may function as inhibitors of PMT and spermidine synthase in vitro and in vivo. In this respect, inhibition of the synthesis of nicotine and nornicotine observed after treating tobacco calli with cyclohexylamine (32) may be interpreted as in vivo inhibition of tobacco PMT by the monoamine inhibitor. As described in the introduction, more and more evidence has been accumulating recently that supports the putrescinemediated pathway for tropane alkaloid biosynthesis. Previous inhibitor-feeding experiments employed a suicide in-

PUTRESCINE N-METHYLTRANSFERASE IN ALKALOID BIOSYNTHESIS

835

hibitor of omithine decarboxylase, which blocked the entire pathway from the starting amino acid (17, 36). A unique advantage of our present feeding experiments is that not only was alkaloid synthesis effectively blocked by the inhibitor of PMT, but accumulation of the enzyme's substrate could also be monitored, thereby substantiating the in vivo target of the inhibitor. Approximately equal degrees of alkaloid decrease and a putrescine increase strongly suggest that PMT, in fact, functions in vivo as the first enzyme that is specific to tropane alkaloid biosynthesis, as well as to nicotine synthesis in tobacco. Molecular cloning of PMT is now underway in our laboratory to study the regulation of the biosynthetic pathways at their entry point.
LITERATURE CITED

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22.
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