Plant Ceil Reports (1981) l: 43-47

Plant Cell Reports © Springer-Verlag 1981

Characterization of a Monoterpene Hydroxylase from Cell Suspension Cultures of C a t h a r a n t h u s roseus (L.) G. Don Vitaly Spitsberg% C a r m i n e J. C o s c i a 1, a n d R o b e r t J. K r u e g e f f Edward A. Doisy Department of Biochemistry, St. Louis University School of Medicine, St. Louis, MO 63104, USA 2 School of Pharmacy, Ferris State College, Big Rapids, MI 49307, USA Received June 27, 1981

C o n d i t i o n s have b e e n e s t a b l i s h e d for the optimization of the specific a c t i v i t y of a m e m b r a n e - b o u n d m o n o t e r p e n e h y d r o x y l a s e from cell s u s p e n s i o n cultures of C a t h a r a n t h u s roseus. In time course studies, the h y d r o x y l a s e and N A D P H - c y t o c h r o m e £ r e d u c t a s e e x h i b i t e d m a x i m a l a c t i v i t i e s 18-20 days after inoculation, i.e., d u r i n g early s t a t i o n a r y phase. By late s t a t i o n a r y phase, enzyme a c t i v i t y had declined. In c o n t r a s t an ~nzyme of priraary "natabo!ism achie~,ed ~ptSm~] specific a c t i v i t y by the 12th day and r e m a i n e d c o n s t a n t through day 26, s y n c h r o n o u s w i t h general growth. E f f e c t s of n u t r i t i o n a l and h o r m o n a l factors on the specific act i v i t y of the h y d r o x y l a s e and cell g r o w t h w e r e evaluated. I n h i b i t o r s of h y d r o x y l a s e a c t i v i t y were also a s s e s s e d in vitro. A soluble form of the m o n o t e r p e n e h y d r o x y l a s e has been d e t e c t e d in c u l t u r e d cells p o s s i bly a f f o r d i n g a u s e f u l source of this enzyme for further p u r i f i c a t i o n .

In the last 20 years the c u l t i v a t i o n of Catha r a n t h u s roseus as callus, c r o w n - g a l l tissue and cell s u s p e n s i o n s has p r o v e n useful for the study of p r i m a r y and s e c o n d a r y m e t a b o l i s m as well as tumor d e v e l o p m e n t (for a review, see Carew, 1975). Nevertheless, the a l k a l o i d p r o f i l e of c u l t u r e d cells (Carew, 1975; Zenk, 1977; Scott et al., 1980; Kurz et el., 1980) is far simpler than that of the o r i g i n a l p l a n t tissue, w h i c h s y n t h e s i z e s an e x t e n s i v e array of indole a l k a l o i d s (Cordell, 1974). If p l a n t tissue culture is to b e c o m e a viable source of s e c o n d a r y me-tabolites, d e t a i l e d studies on the o p t i m i z a t i o n of c o n d i t i o n s for their p r o d u c t i o n are necessary. One a p p r o a c h to achieve this goal i n v o l v e s the e n h a n c e m e n t of a c t i v i t i e s of r e l e v a n t r e g u l a t o r y enzymes. A number of the enzymes i m p l i c a t e d in the indole a l k a l o i d p a t h w a y have b e e n i s o l a t e d from C. r o s e u s and its cells in culture (Madyastha and Coscia, 1979a; Zenk, 1980; M i z u k a m i et al., 1979)~ One of these, a monot e r p e n e hydroxylase, c a t a l y z e s a p o s s i b l e r e g u l a t o r y step of the i s o p r e n o i d b r a n c h of the a l k a l o i d biogenetic p a t h w a y (McFarlane et al., 1975). Both a c y c l i c m o n o t e r p e n e alcohols, g e r a n i o l and nerol, are s u b s t r a t e s for this enzyme, b e i n g c o n v e r t e d to 10-hydroxy derivatives. E v i d e n c e f r o m studies w i t h C. roseus s e e d l i n g s (Madyastha et al., 1976) suggest that this n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e d e p e n d e n t m o n o o x y g e n a s e is a m u l t i e n z y m e complex. On the basis of its e x q u i s i t e s e n s i t i v i t y to c y t o c h r o m e c as well as r e c o n s t i t u t i o n studies (Madyastha and Coscia, !979b) an N A D P H - c y t o c h r o m e c r e d u c t a s e is c o n s i d e r e d to be a c o m p o n e n t of this system.

In this p a p e r we report on the time course of a p p e a r a n c e and the p a r t i a l c h a r a c t e r i z a t i o n of the hydroxylase from C. roseus cell s u s p e n s i o n cultures. The enzyme was found to attain its h i g h e s t specific a c t i v i t i e s in the early s t a t i o n a r y phase of the g r o w t h curve and then to decline. Unlike its c o u n t e r p a r t in seedlings, a s i g n i f i c a n t f r a c t i o n o f the enzyme appears to be p r e s e n t in a soluble or loosely b o u n d form.

_MATERIALS A N D M E T H O D S Tissue Source and Maintenance. The source of suspension culture tissue used in this study was C a t h a r a n thus roseus (L.) G. Don callus line PRLI, i n i t i a t e d in 1972 (Carew and Krueger, 1977). The basal m e d i u m (B5) u t i l i z e d for callus and cell s u s p e n s i o n culture was d e v e l o p e d by G a m b o r g (1970). P r o c e d u r e s for m a i n t e nance of b o t h callus and s u s p e n s i o n c u l t u r e s have b e e n p r e v i o u s l y d e s c r i b e d (Carew and Krueger, 1977). M e t h o d s of Cultivation. Callus tissue (24 f 2 days old) was u s e d to initiate s u s p e n s i o n cultures. The latter w e r e g r o w n in 500 ml c o t t o n - s t o p p e r e d Erlenmeyer flasks c o n t a i n i n g i00 ml of medium. Flasks were shaken c o n t i n u o u s l y on rotary shakers at 130 + 5 rpm at 24 + l°C. P r o c e d u r e s for the i n c l u s i o n of add i t i o n a l m e d i u m constituents., i.e., h o r m o n e s and p r e cursors have b e e n p r e v i o u s l y r e p o r t e d (Krueger and Carew, 1978). Levels r e p o r t e d always r e p r e s e n t final m e d i u m concentration. Unless i n d i c a t e d otherwise, all a d d i t i v e s w e r e i n t r o d u c e d at the time of inoculation. G r o w t h indices (GI) were c a l c u l a t e d using the formula: GI : (harvest w e i g h t - i n o c u l u m wt) x 1 0 / ( i n o c u l u m wt x age in days). GI for control tissue r a n g e d from 1.0 to 3.0. P r~aration of Microsomes. All steps were p e r f o r m e d at 0 - 4°C. Cells were added to 2 - 3 v o l u m e s of the f o l l o w i n g e x t r a c t i o n medium: 220 m M D-Mannitol, 70 ~ 4 sucrose, 20 m M p o t a s s i u m p h o s p h a t e or T r i s - H C l buffer, p H 7.4 - 7.5. Cells w e r e then d i s r u p t e d in a glass (Wheaton) h o m o g e n i z e r by five strokes of a mot o r - d r i v e n teflon p e s t l e m a i n t a i n e d at i000 rpm. Cellular debris was r e m o v e d by c e n t r i f u g a t i o n at 1,000 g for i0 min. The s u p e r n a t a n t was then s e d i m e n t e d at i0,000 g for i0 min to o b t a i n a crude m i t o c h o n d r i a l pellet. The s u p e r n a t a n t was r e c e n t r i f u g e d at 105,000 for 75 m i n to o b t a i n the m i c r o s o m a l pellet. Sedim e n t e d m i c r o s o m e s w e r e r e s u s p e n d e d in a s u f f i c i e n t volume of 50 m H p o t a s s i u m p h o s p h a t e buffer, p H 7.5, i0 - 15% glycerol, 1 m M E D T A and 0.i m M DTT, to a f f o r d

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X GERANIOL HYDROXYLASE NADPH-cyt c REDUCTASE • GLUCOSE-6-P D E H Y D R O G E N A S E A CATALASE O GI I I I I I I 16 18 20 22 24 26

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28

DAYS

Fig. 1 Age D e p e n d e n c y of Enzyme Specific A c t i v i t i e s of C. roseus Cell S u s p e n s i o n Cultures. A f t e r i n o c u l a t i o n w i t h callus tissue, cultures w e r e grown for 26 days. M i c r o s o m e s were i s o l a t e d and a s s a y e d for enzyrae a c t i v i t i e s every 2 days b e g i n n i n g on day i0. A unit represents: 5 n m o l / h r / m g p r o t e i n for g e r a n i o l hydroxylase; 25 n m o l / m i n / m g p r o t e i n for NADPH-cyt. £ reductase; 0.25 5 0 D 3 4 0 n m / m i n / m g p r o t e i n for g l u c o s e - 6 - p h o s p h a t e dehydrogenase; 25 n m o l / m i n / m g p r o t e i n for catalase; and a value of 1.0 for GI. See "Methods" for a d d i t i o n a l details.

a final p r o t e i n c o n c e n t r a t i o n of 3 - 5 mg/ml. Mic r o s o m e s w e r e a s s a y e d either i m m e d i a t e l y or stored under liquid n i t r o g e n under w h i c h conditions hydroxylase a c t i v i t y was stable for several months. Enzyme sitive 1976), 1955) sayed

Assays. G e r a n i o l hydroxylase, cyanide insenN A D P H - c y t o c h r o m e £ r e d u c t a s e (Madyastha et al., g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (DeMoss, and c a t a l a s e (Beers and Sizer, 1952) were asusing p r e v i o u s l y d e s c r i b e d procedures.

P r o t e i n Determination. A m o d i f i e d Lowry assay was used for p r o t e i n e s t i m a t i o n (0hnishi and Barr, 1978).

RESULTS Before studies on the c h a r a c t e r i z a t i o n of the m o n o t e r p e n e h y d r o x y l a s e from C. roseus s u s p e n s i o n cultures w e r e initiated, it was a p p r o p r i a t e to a t t e m p t to e s t a b l i s h c u l t u r e c o n d i t i o n s w h i c h give o p t i m a l enzyme activity. F r o m p r e l i m i n a r y e x p e r i m e n t s it was a p p a r e n t that u p o n i n t r o d u c t i o n of a callus i n o c u l u m into liquid culture, a lag or i n d u c t i o n p e r i o d occurred d u r i n g the first week. This was f o l l o w e d by e x p o n e n t i a l g r o w t h w h i c h a p p e a r e d to level off by the second week, the cells e n t e r i n g s t a t i o n a r y p h a s e at this time. E x a m i n a t i o n of specific a c t i v i t i e s of enzymes of p r i m a r y and secondary m e t a b o l i s m in a p e r i o d from i0 through 26 days (Fig. i) r e v e a l e d a general p a r a b o l i c rise synchronous w i t h growth. However, the enzymes a s s a y e d a p p e a r e d to reach optimal a c t i v i t y at d i s t i n c t l y d i f f e r e n t time intervals. Thus an enzyme of p r i m a r y metabolism, g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , a p p e a r e d to achieve m a x i m a l ac-

tivities

before

the other

enzymes

tested.

The m i c r o s o m a l m o n o t e r p e n e h y d r o x y l a s e and NADPHcytochrome c reductase exhibited optimal activities c o n s i s t e n t l y d u r i n g the early s t a t i o n a r y p h a s e (18 20 days) in four time course e x p e r i m e n t s and in each e x p e r i m e n t a s i g n i f i c a n t d e c l i n e s u b s e q u e n t l y occurred. This d e c r e a s e was not o b s e r v e d for g l u c o s e - 6 phosphate dehydrogenase. Interestingly, the a c t i v i t y of the c a t a l a s e in the m i c r o s o m a l f r a c t i o n appears to reach a m a x i m u m in the late s t a t i o n a r y p h a s e period, i.e., after the hydroxylase. In all subsequent invest i g a t i o n s of the m o n o t e r p e n e h y d r o x y l a s e and reductase, 18 - 20 day old cells were used. In p r e v i o u s studies g r o w t h and a l k a l o i d p r o d u c tion were a d o p t e d as c r i t e r i a for m e d i u m s e l e c t i o n (Carew and Krueger, 1977; Zenk et al., 1977). In this research, changes in the levels of m e d i u m nutrients, g r o w t h factors and a l k a l o i d p r e c u r s o r s w e r e e v a l u a t e d for their effect on the a c t i v i t i e s of m o n o terpene h y d r o x y l a s e and/or N A D P H - c y t o c h r o m e c reductase. Since cell suspension cultures were d e r i v e d from callus i n o c u l u m of v a r i o u s w e i g h t s and grown for slightly d i f f e r e n t lengths of time, g r o w t h was m o r e a c c u r a t e l y r e f l e c t e d by GI. These v a r i a b l e s are included in the e q u a t i o n u s e d to c a l c u l a t e GI. The m e d i a c o n s t i t u e n t s that w e r e v a r i e d were the m a j o r sources of carbon and nitrogen. W h e n the level of m e d i u m NH4 + was e l e v a t e d by i n c r e a s i n g the c o n c e n t r a t i o n of a m m o n i u m sulfate, 1.2, 1.5 and 2 fold, total protein, GI and the specific a c t i v i t i e s of both the g e r a n i o l h y d r o x y l a s e and the r e d u c t a s e w e r e e s s e n t i a l l y the same. In a 3 - 5 w e e k long culture

45

l

Table Effects

of Inhibitors

Inhibitor

Conc. mM

Cytochrome ~ Ellipticine 1-Phenylimidazole

Metyrapone SKF525A Nordihydroguaiaretic Cumene peroxide

of G e r a n i o l

acid

0.008 0.20 0.10 0.20 0.40 0.20 1.00 0.i0 1.00

I

I

I

I

I

F

i

Hydroxylase

0.6

% Activity

50 87 i18 93 38 95 60 70 81

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