i

Plant Cell Reports (1985) 4:216-219

Plant Cell Reports © Springer-Verlag 1985

Partial purification and characterization of a N A D P H dependent tetrahydroalstonine synthase from C a t h a r a n t h u s roseus cell suspension cultures * T. Hemscheidt and M. H. Zenk Lehrstuhl ~ r Pharmazeutische Biologie, Univ6rsit~it Mflnchen, Karlstrasse 29, D-8000 Mt~nchen 2, FRG Received July 8, 1985 - Communicated by K. Hahlbrock

ABSTRACT A new enzyme was d i s c o v e r e d w h i c h s p e c i f ically h y d r o g e n a t e s the i m i n i u m form o f c a t h e n a m i n e at p o s i t i o n 21 to y i e l d t h e heteroyohimbine alkaloid tetrahydroalstonine. The enzyme was p a r t i a l l y purified (35-fold) from C a t h a r a n t h u s roseus c e l l s u s p e n s i o n cultures. I t was shown to use e x c l u s i v e l y NADPH as r e d u c t a n t , t h e pH optimum i s at 6 . 6 , the t e m p e r a t u r e optimum at 30°C, the h a l f l i f e o f t h e s o l u b l e enzyme p r e p a r a t i o n is 26 min at 37°C, and t h e m o l e c u l a r w e i g h t i s 81 000 + 3%. E v i d e n c e i s p r e s e n t e d f o r the o c c u r r e n c e o f two d i s t i n c t and d i f f e r e n t c a t h e n a m i n e r e d u c t a s e s , one r e d u c i n g t h e i m i n i u m form of t h i s c e n t r a l i n t e r m e d i a t e to give tetrahydroalstonine, the o t h e r one r e d u c i n g c a t h e n a m i n e to y i e l d a j m a l i c i n e . Tetrahydroalstonine s y n t h a s e was p r e s e n t in c e l l s u s p e n s i o n c u l t u r e s o f C. o v a l i s , C. r o s e u s , P i c r a l i m a n i t i d a , ~ h a z y a stricta, a-nd~ca herbacea. INTRODUCTION Tetrahydroalstonine, a member of the group of h e t e r o y o h i m b i n e t y p e a l k a l o i d s , is commonly f o u n d in i n d o l e a l k a l o i d c o n t a i n i n g p l a n t s p e c i e s of the f a m i l i e s Apocynaceae and Rubiaceae (Hesse 1965; 1 9 6 8 ) . I t s e n z y m a t i c f o r m a t i o n has been observed in crude c e l l - f r e e e x t r a c t s of C a t h a r a n t h u s roseus i n the p r e s e n c e of t r y p t a m i n e , ~ganin, and reduced p y r i d i n e n u c l e o t i d e s (StOckigt et al., 1976). F u r t h e r m o r e , c a t h e n a m i n e and i t s i m i n i u m form have been e s t a b l i s h e d as p r e c u r s o r s o f t e t r a h y d r o alstonine (StOckigt et al., 1977; H e i n s t e i n et al., 1980). This r e p o r t d e s c r i b e s the p a r t i a l purifi c a t i o n and c h a r a c t e r i z a t i o n of t e t r a h y d r o a l s t o n i n e s y n t h a s e , an enzyme s p e c i f i c a l l y i n v o l v e d in t h e b i o s y n t h e s i s o f t h i s heteroyohimbine alkaloid. MATERIAL and METHODS Cell

Cultures

A c e l l l i n e o f C. r o s e u s was s e l e c t e d ( s t r a i n 106, B. Deus-Neumann a N M.H. Zenk, un-

p u b l i s h e d ) w h i c h produced t e t r a h y d r o a l s t o n i n e and no o t h e r h e t e r o y o h i m b i n e a l k a l o i d . This s t r a i n and s t r a i n SR 28 (B. Deus-Neumann and M,H. Zenk, 1984) were grown in L i n s m a i e r and Skoog (1965) medium ( I - i Erlenmeyer flasks containing, 300 ml medium) at 25°C f o r 7 days w i t h s h a k i n g at 100 rpm. C e l l c u l t u r e s of C, o v a l i s , Picralima nitida, Rhazya s t r i c t a , ~ d ~ V i n c a herbacea were g r o w n ~ t h e sage c o n d ] t - i o n s . In ~ h case c e l l s were h a r v e s t e d by f i l t r a t i o n , t h e t i s s u e was f r o z e n in l i q u i d N2, and was t h e n s t o r e d at -18°C u n t i l used. Enzyme P r e p a r a t i o n To d e e p - f r o z e n p l a n t c e l l s was added a double volume of 0.1 M b o r a t e b u f f e r , pH 7 . 6 , c o n t a i n i n g 20 mM B - m e r c a p t o e t h a n o l . The m i x t u r e was s t i r r e d u n t i l t h e t i s s u e was t h a w e d , and s u b s e q u e n t l y p r e s s e d t h r o u g h cheese-cloth. The l i q u i d was c e n t r i f u g e d f o r 20 min at 27 000 x g. (NH4)2SO 4 was added to t h e c l e a r s u p e r n a t a n t to 40% s a t u r a t i o n . The precipitated p r o t e i n was c e n t r i f u g e d o f f ; t h e n (NH4)2SO 4 was added to t h e s u p e r n a t a n t to 70% s a t u r a t i o n . After centrifugation the s e d i m e n t was t a k e n up in 0.05 M KP042b u f f e r , pH 7 . 0 . T h i s p r o t e i n s o l u t i o n (63 ml) was added to t h e top o f an U l t r o g e l AcA-44 column (5 x 95 cm; gel bed volume 1865 ml; speed o f e l u t i o n : 1.5 m l / m i n ; e l u t i o n b u f f e r 25 mM KP042- b u f f e r , pH 7 . 0 ; f r a c t i o n size 20 m l ) . The enzyme was f o u n d in f r a c t i o n s 4 2 - 6 2 . These f r a c t i o n s were p o o l e d , concentrated by ammonium s u l f a t e p r e c i p i t a t i o n (70% saturation) and s u b s e q u e n t l y d e s a l t e d by c h r o m a t o g r a p h y on a Sephadex G-25 column ( 2 . 3 x 8 cm) p r e v i o u s l y e q u i l i b r a t e d with 20 mM KPO,2- b u f f e r , pH 7 . 0 , An 8 ml s~mple o f s a l t - f r e e s o l u t i o n was a p p l i e d to a column ( 2 . 3 x 8 cm) c o n t a i n i n g M a t r e x g r e e n - g e l ( A m i c o n ) . The p r o t e i n was e l u t e d w i t h 20 mM KP042- b u f f e r (pH 7 . 0 ) , e l u t i o n speed 0.15 m I / m i n , f r a c t i o n s i z e 3 ml. Under t h e s e c o n d i t i o n s t h e enzyme appeared in f r a c t i o n s 15-20. A t t e m p t s to f u r t h e r p u r i f y t h e enzyme f a i l e d . For i o n - e x c h a n g e c h r o m a t o g r a p h y an 8-10 ml sample, p r e purified by gel f i l t r a t i o n on AcA-44 and desalted in 10 mM KP042- b u f f e r (pH 7 . 0 ) was a p p l i e d {o a D E A E - c e l l d l o s e column (Whatman DE 52, 2.3 x 10 cm), w h i c h was washed w i t h

* Dedicated to Prof. Dr. Franz Lingens on the occasion of his 60th birthday Offprint requests to: T. Hemscheidt

217

Purification

step

Volume (ml)

Total protein

(mg) Crude e x t r a c t

Specific activity (pkat/mg)

Recovery (%)

Purification -fold

I

1210

1890

47

846

0.93

100

235

174

3.86

85

18

11

(NH4)2SO 4 cut 40-70% Gel f i l t r a t i o n Dye-ligand

(AcA-44)

chromatography

(Matrex green) Table I

32.4

Typical purification procedure for tetrahydroalstonine from C a t h a r a n t h u s roseus c e l l s u s p e n s i o n c u l t u r e s .

10 mM KP042-, pH 7 . 0 , and t h e n e l u t e d w i t h a l i n e a r g r a d i e n t o f KCI ( 0 - 0 . 2 M) in t h i s b u f f e r ( 0 . 5 m l / m i n , 5 ml f r a c t i o n size). P r o t e i n was q u a n t i f i e d a c c o r d i n g to B r a d f o r d ( 1 9 7 6 ) . The t y p i c a l p u r i f i c a t i o n procedure f o r t h e enzyme is shown in T a b l e I . M o l e c u l a r Weight

3.9

Determination

The m o l e c u l a r w e i g h t o f t h e p u r i f i e d synthase was d e t e r m i n e d by g e l f i l t r a t i o n on a c a l i b r a t e d Sephadex G-IO0 s u p e r f i n e column. The column 132.5 m i _ ( I . 5 x 75 cm) e q u i l i b r a t e d w i t h 50 mM KP04Z- b u f f e r a t pH 7 . 0 , was e l u t e d a t a f l o w r a t e o f 0.25 m l / m i n in fractions o f 2 mI. Sample volume was I mi. The column was c a l i b r a t e d u s i n g t h e Combithek (Boehringer) standard protein mixture. Ferritin (Mr 45 000) was used f o r the determina t i o n o f t h e v o i d volume o f t h e column. P r o t e i n e l u t i o n was m o n i t o r e d by the absorbance a t 280 nm. The r e s u l t s are e x p r e s s e d as Stokes r a d i i . Enzyme Assay Since c a t h e n a m i n e is o n l y s l i g h t l y soluble in w a t e r , t h e s u b s t r a t e was added as a s o l u t i o n in D i m e t h y l f o r m a m i d e (2 m g / m l ) . Ten pl o f t h i s s o l u t i o n were added t o 20 pMol KP042- b u f f e r , pH 6 . 6 , 500 nMol NADPH and p r o t e i n s o l u t i o n t o make a f i n a l volume o f 210 pl (5% DMF v / v ) . A f t e r i n c u b a t i o n f o r 20 min a t 30°C t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 20 pl I M Na2CO3 s o l u t i o n (final pH 10). The m i x t u r e s were shaken f o r 30 sec ( E p p e n d o r f m i x e r ) and t h e n 20 ~I a l i q u o t s were i n j e c t e d o n t o a HPLC column. One k a t o f enzyme is d e f i n e d as t h a t amount o f enzyme which reduces I mole c a t h e n a m i n e a t pH 6 . 6 , 30°C w i t h a s u b s t r a t e c o n c e n t r a t i o n o f 285 pM a t a NADPH c o n c e n t r a t i o n o f 2.5 mM. For t h e d e t e r m i n a t i o n o f k i n e t i c c o n s t a n t s a m o d i f i e d assay was e m p l o y e d . The NADPH c o n c e n t r a t i o n was v a r i e d in f o u r s t e p s ( 0 . 1 4 5 ; 0 . 2 1 5 ; 0 . 3 5 5 ; 0.43 mM), at each o f f o u r f i x e d cathenamine concentrations (0.1; 0.14; 0.25; 0 . 4 mM). The t o t a l volume was 0.5 ml containing 50 pMol KP042- b u f f e r , pH 6 . 6 . Samples o f 50 pl were a n a l y s e d in t r i p l i c a t e for alkaloid c o n t e n t by HPLC. S t r i c t o s i d i n e s y n t h a s e was assayed a c c o r d i n g t o T r e i m e r and Zenk ( 1 9 7 9 ) , and s p e c i f i c s t r i c t o s i d i n e g l u c o s i d a s e s were assayed a c c o r d i n g t o Hemscheidt and Zenk (1980).

45

34

synthase

Separation of Alkaloids

by HPLC

The assay used by F e l i x e t a i . (1981) was a p p l i e d . A Merck Li Chro C a r t RP 18 10 pm (250 x 4 mm) column and a Vydac-201 RP 30-44 pm p r e c o l u m n (65 x 4 mm) were employed. As s o l v e n t I% ( N H 4 ) 2 C O 3 : a c e t o n i t r i l e 45:55 was used. Speed o f e l u t i o n was I m l / m i n , w i t h d e t e c t i o n a t 280 nm. Under t h e s e c o n d i t i o n s , 0.025 pg t e t r a h y d r o a l s t o n i n e c o u l d be detected. S t a n d a r d d e v i a t i o n up t o 0.5 pg a l k a l o i d was + 10%. The a l k a l o i d s e l u t e d from t h e column Tn t h e f o l l o w i n g o r d e r : a j m a l i c i n e (10.25 min); 19-epi-ajmalicine (11.39 min); cathenamine (15.21 m i n ) ; t e t r a h y d r o a l s t o n i n e (17.34 min). RESULTS and DISCUSSION The p r e s e n c e o f t h e NADPH-dependent t e t r a h y d r o a l s t o n i n e s y n t h a s e was d e m o n s t r a t e d in c e l l c u l t u r e s d e r i v e d from f o u r members o f t h e p l a n t f a m i l y A p o c y n a c e a e : C. r o s e u s , C. o v a l i s , Rhazya s t r i c t a , and Vinca h e r b a c e a ( T a b l e 2 ) . C. roseus s t r a i n 106 p r o v e d t o be t h e b e s t s o u r c e o f The enzyme. F i g . I shows the e l u t i o n p r o f i l e o f t h e crude p r o t e i n e x t r a c t from t h i s c u l t u r e ( r e d i s s o l v e d (NH4)2SO 4 p r e c i p i t a t e a f t e r gel f i l t r a t i o n ) . The a c t i v i t i e s o f t h r e e enzymes - s t r i c t o s i d i n e s y n t h a s e ( T r e i m e r and Zenk, 19791, specific (strictosidine) g l u c o s i d a s e (Hems c h e i d t and Zenk, 1980) and t e t r a h y d r o alstonine synthase are p l o t t e d vs e l u t i o n volume. The p u r e s t enzyme p r e p a r a t i o n obtaine~ was e n r i c h e d 3 3 - f o l d and i t c o n t a i n e d . 4 5 % o f the t o t a l a c t i v i t y p r e s e n t in t h e crude extract. No o t h e r enzymes i n v o l v e d in i n d o l e a l k a l o i d b i o s y n t h e s i s c o u l d be detected~ in the p u r i f i e d preparation. The 3 3 - f o l d e n r i c h e d p r o t e i n s o l u t i o n was used t o d e t e r m i n e t h e enzyme's p h y s i c a l p r o p e r t i e s and t o examine i t s c a t a l y t i c ; .... activity. The m o l e c u l a r w e i g h t was determined by gel f i l t r a t i o n t o be 81 ± 2.4 kD, asSuming a g l o b u l a r shape f o r t h e m o l e c u l e . When t h e enzyme was i n c u b a t e d w i t h c a t h e n a m i n e , .the o n l y r e a c t i o n p r o d u c t o b s e r v e d by HPLC was tetrahydroalstonine. Only NADPH + H+ s e r v e d as a r e d u c i n g c o f a c t o r f o r t h e r e a c t i o n . I t is e x p e c t e d t h a t t h i s enzyme d o n a t e s t h e h y d r i d e o f NADPH t o C-21 o f c a t h e n a m i n e in ~ - p o s i t i o n as d e m o n s t r a t e d by S t b c k i g t e t a l . ( 1 9 8 3 ) . A b s o l u t e l y no c o n v ' e r s i o n o'f

218 Plant material

Family

Catharanthus

ovalis

Apocynaceae

7

0.12

roseus

Apocynaceae

7

0.98

Apocynaceae

7

0.51

Rhazya s t r i c t a

Apocynaceae

18

0.64

V i n c a herbacea

Apocynaceae

7

0.10

nitida

Beta v u l g a r i s

Chenopodiaceae

7

0

Malus d o m e s t i c a

Rosaceae

7

0

Solanum m a r g i n a t u m

Solanaceae

7

0

Table 2

Survey of d i s t r i b u t i o n of tetrahydroalstonine s y n t h a s e in c u l t u r e s of different i n d o l e a l k a l o i d c o n t a i n i n g p l a n t s and c o n t r o l t i s s u e .

/

-o

I.~ 2 ;~

i I•

" , ~ 17

,, i ,'7 A

2b

io

1

, ~ 6r9 "

.,

;

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i'

u

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~o

16o

I~

Fraction N o

Fig.

Enzyme a c t i v i t y (pkat/mg protein)

Catharanthus Picralima

u

Age of c e l l culture (days)

I

t u r n e d o u t t h a t t h e enzyme most l i k e l y follows "rapid equilibrium" ordered BiBi mechanism (Segal e t a l . , 1952) u n d e r t h e reaction conditions described. In t h i s mechanism t h e measured r e a c t i o n r a t e i s i n d e p e n d e n t o f t h e KM v a l u e o f t h e enzyme for the first binding substrate since the e n z y m e - s u b s t r a t e complex r e a r r a n g e s v e r y f a s t to a s p e c i e s t h a t can b i n d t h e second s u b s t r a t e . The KM v a l u e f o r c a t h e n a m i n e u n d e r the given conditions (pH 6 . 6 , 30°C, 5% DMF) was 62 ~M. Complete i n h i b i t i o n of enzyme activity was o b s e r v e d both w i t h t h i o l r e a g e n t s and w i t h m e t a l ions at t h e f o l l o w i n g concentrations: p-(OH)mercuribenzoate (100 pM), i o d o a c e t a m i d e (10 mMl~ 2 , 2 ' - d i ~ i o -4,4l-dinitrobisbenzoic acid ( mM), Fe , Cu++, Hg++, and Z n + + ( a l l 10 mM). In a l l cases i n h i b i t i o n was i r r e v e r s i b l e .

Elution p r o f i l e of tetrahydroalstonine synthase a c t i v i t y from an Ultrogel AcA-44 gel f i l t r a t i o n column using 25 mM potassium phosphate buffer, pH 7.0 as e l u t i o n solvent. The applied protein solution had been p r e p u r i f i e d by an ammonium sulfate f r a c t i o n a t i o n (40-70%) as given in Material and Methods.

100-

•>- 80c a t h e n a m i n e c o u l d be o b s e r v e d when t h i s n u c l e o t i d e was r e p l a c e d by NADH. The dependence o f enzyme a c t i v i t y on H" c o n c e n t r a t i o n was examined in t h e range from pH 4 - 9 . F i g . 2 shows t h a t t h e c a t a l y t i c activity has a sharp optimum at pH 6 . 6 . S i m i l a r v a l u e s have been f o u n d f o r t h e two o t h e r known h e t e r o y o h i m b i n e biosynthetic enzymes s t r i c t o s i d i n e synthase ( 6 . 5 , T r e i m e r and Zenk, 1979) and s t r i c t o s i d i n e g l u c o s i d a s e ( 6 . 3 , Hemscheidt and Zenk, 1 9 8 0 ) . Maximal c a t a l y t i c activity of the enzyme was o b s e r v e d at 30°C. Using t h e i n i t i a l velocities measured f o r r e a c t i o n s in t h e range 5 - 3 0 ° C , a v a l u e of 1.65 k J / m o l was c a l c u l a t e d for the free energy of activation. This c o r r e s p o n d s to a QIO v a l u e o f 1 . 2 5 , w h i c h i s substantially lower than typically observed in enzyme r e a c t i o n s . The r a t e o f t h e r m a l inactivation o f t h e enzyme was measured at 37°C; at t h i s t e m p e r a t u r e t h e enzyme a c t i v i t y has a h a l f - l i f e o f 26 min. On a n a l y s i s o f t h e i n i t i a l by e s t a b l i s h e d p r o c e d u r e s

rate experiments (Fromm, 1975) i t

E 60N ¢-

40" o¢

20"

7

8

9

pH Fig.

2

The e f f e c t of pH on tetrahydroalstonine synthase a c t i v i t y (2.3 p k a t / t e s t ) . The assay was performed in the presence of 0.1M (e - e) c i t r a t e phosphate; (A - A) acetate; (x - x) phosphate and (o - o) tris-HCl b u f f e r .

219 No i n h i b i t i o n was o b s e r v e d i n t h e p r e s e n c e of EDTA, Co ++ , Mn++ or Mg++, and none o f t h e m e t a l i o n s t e s t e d enhanced t h e r a t e o f reaction. The r e a c t i o n i s i r r e v e r s i b l e , when t e t r a h y d r o a l s t o n i n e and the+enzyme are i n c u b a t e d i n t h e p r e s e n c e o f NAD or NADP+ o v e r a wide range o f t i m e , c o f a c t o r c o n c e n t r a t i o n s and pH v a l u e s , no c a t h e n a m i n e nor reduced p y r i d i n e n u c l e o t i d e c o u l d be d e t e c t ed i n t h e m i x t u r e . When p r o t e i n e x t r a c t s o f c e I ] c u l t u r e s were prepurified by (NH4)2SO 4 f r a c t i o n a t i o n and gel f i l t r a t i o n (Sephadex G - 2 5 ) , and t h e n assayed f o r t e t r a h y d r o a l s t o n i n e synthase activity, o n l y members o f t h e Apocynaceae f a m i l y gave p o s i t i v e tests (Table 2). Contro| samples t a k e n from c u l t u r e s o f p l a n t s known not t o p r o d u c e i n d o l e a ] k a l o i d s ( e . g . Beta vulgaris, Malus d o m e s t i c a , and Solanum m a r g i n a t u m ) c o n t a i n e d no measura--b--~e a c t i v i t y . The t e t r a h y d r o a I s t o n i n e synthase obtained from a c e i l - f r e e p r e p a r a t i o n o f C. r o s e u s s t r a i n SR 28 (Deus-Neumann and Z ~ k ~ ) and p r e p u r i f i e d by gel f i ] t r a t i o n on U I t r o gel AcA-44 was s e p a r a t e d from a second c a t h e n a m i n e r e d u c t a s e by c h r o m a t o g r a p h y on D E A E - c e I i u l o s e . On e l u t i o n w i t h a l i n e a r KCI g r a d i e n t one enzyme, f o r m i n g a j m a l i c i n e and 19-epi-ajmalicine, e I u t e d at 0 . 0 5 M KC! and THA s y n t h a s e was l i b e r a t e d at 0.1 M KCI ( F i g . 3 ) . Thus we r e p o r t f o r t h e f i r s t time t h a t two s e p a r a t e r e d u c t a s e s c a t a l y z e t h e formation of these stereoisomeric indole alkaloids.

I

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05

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i

,- to

1

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il

.",

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20

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02 i i

0.1 %.

.

.

.....

30

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50

Fraction-No. Fig.

3

E1ution p r o f i l e of cathenamine reducing enzymes from a DEAE-cellulose co]umn using a l i n e a r gradient of KCI (0.-0.2 M) in 10 mM KPOA2buffe~ pH 7.0. The ce11-free extract from Catharanthus roseus had been p r e p u r i f i e d by ge] f i l t r a t i o n on Ultrogel AcA-44 (see Material and Methods).

REFERENCES

,

N

H H3 ..,,,'~h.."~,,,C H" Cathcnamin¢ [Iminium form)

NADPH÷H* ~

NADP* ~,

~,,,,H CH HY ~" 3 Tetrohydroolstonin¢

A c k n o w l e d g e m e n t s : F i n a n c i a l a s s i s t a n c e from t h e Deutsche F o r s c h u n g s g e m e i n s c h a f t , Bonn, SFB 145 and from Fonds der Chemischen I n dustrie is gratefully a c k n o w l e d g e d . We a l s o t h a n k Dr. James K i l g o r e f o r h i s l i n g u i s t i c help with the English text.

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