Regulation of Isopentenoid Metabolism - American Chemical Society

Chapter 3

Natural Rubber Biosynthesis A Branch of the Isoprenoid Pathway in Plants

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch003

Katrina Cornish Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710

Natural rubber is a plant product in high demand in the United States. Chemically, natural rubber is a high molecular weight hydrocarbon polymer largely composed of isoprene monomers linked, in a head-to-tail fashion, to form long chains of cis-1,4-polyisoprene. Simply stated, the longer the molecule, the better the performance characteristics of the rubber. Synthetic rubber cannot substitute for natural rubber in applications that require high elasticity, resilience and minimal heat build-up (e.g., high performance tires, latex gloves). Natural rubber is often blended with synthetic forms to confer desirable high performance characteristics. Synthetic rubber is derived from petroleum products and so is a non-renewable resource; natural rubber should be available indefinitely from renewable plant sources. At present, the United States imports over 1,000,000 tons of natural rubber each year, at a cost of approximately $1,000,000,000, to support its commercial and defense needs. Natural rubber constitutes over 40% of the total amount of both natural and synthetic rubber used and is expected to increase its market share substantially over the next few years, leading to an even greater demand. However, virtually a l l natural rubber is obtained by tapping the Brazilian rubber tree (Hevea brasiliensis), a tropical species. Even i f H. brasiliensis could be grown in some developed countries, the cultivation and harvesting costs would be prohibitive. Consequently, developed countries, and temperate regions of the world, are wholly dependent on nondomestic sources of this vital raw material. The capacity for rapid expansion of domestic natural rubber production would protect the United States against emergency shortages induced by crop failure in the H. brasiliensis plantations or by changing political climes. The strategic necessity of rubber is, This chapter not subject to U.S. copyright Published 1992 American Chemical Society

In Regulation of Isopentenoid Metabolism; Nes, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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of i t s e l f , s u f f i c i e n t i n c e n t i v e t o develop a domestic r u b b e r - p r o d u c t i o n s y s t e m , even i f i t l a c k e d c o m m e r c i a l competitiveness at the time of i t s completion. N e v e r t h e l e s s , c r o p c o s t s w i l l c e r t a i n l y r i s e as t h e producing countries develop further. The p r o s p e c t s o f commercial competitiveness of domestic rubber w i l l s t e a d i l y i m p r o v e as n a t u r a l r u b b e r p r i c e s i n c r e a s e . Our r e s e a r c h i s d i r e c t e d t o w a r d s t h e d e v e l o p m e n t o f a domestic n a t u r a l rubber supply f o r the United S t a t e s . To a c h i e v e t h i s g o a l we have d e c i d e d upon a b i o t e c h n o l o g i c a l a p p r o a c h : t o t h i s end we n e e d t o i d e n t i f y and i s o l a t e t h e enzymes and g e n e s r e s p o n s i b l e for the c i s - 1 4 - p o l y m e r i z a t i o n of isoprene unique t o rubber-producing plants. Of c o u r s e , we must a l s o c l a r i f y t h e b i o c h e m i s t r y o f r u b b e r f o r m a t i o n and u n d e r s t a n d t h e i n v i v o f u n c t i o n i n g o f t h e enzymes and g e n e s i n v o l v e d i n n a t u r a l rubber b i o s y n t h e s i s . Once t h i s i s d o n e , i t s h o u l d be p o s s i b l e t o i s o l a t e , t h e n i n s e r t and e x p r e s s , t h e a p p r o p r i a t e genes i n annual p l a n t s and, p e r h a p s , i n m i c r o - o r g a n i s m s , and t h e n o p t i m i z e t h e s e s y s t e m s t o produce h i g h q u a l i t y n a t u r a l rubber.


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Sources o f N a t u r a l Rubber N a t u r a l r u b b e r i s s y n t h e s i z e d and a c c u m u l a t e d i n more t h a n 2 , 0 0 0 s p e c i e s o f p l a n t s f r o m a b o u t 300 g e n e r a , as w e l l a s i n a t l e a s t two f u n g a l g e n e r a ( 4 , 6 ) . The h i g h q u a l i t y r u b b e r p r o d u c e d by H . b r a s i l i e n s i s h a s a mean m o l e c u l a r weight o f about 1,500,000 Da. Parthenium argentatum A . Gray (guayule) a l s o produces h i g h m o l e c u l a r weight rubber. H o w e v e r , most r u b b e r - p r o d u c i n g p l a n t s make much s h o r t e r r u b b e r c h a i n s o f < 5 0 , 0 0 0 D a . R u b b e r y i e l d c a n a l s o v a r y g r e a t l y from s p e c i e s t o s p e c i e s . T h i s w i d e r a n g e o f a v a i l a b l e m a t e r i a l c a n be e x p l o i t e d when a d d r e s s i n g d i f f e r e n t a s p e c t s o f t h e d e v e l o p m e n t o f a domestic rubber resource. A h i g h l y developed p l a n t such as fl. b r a s i l i e n s i s t h a t forms i t s r u b b e r i n a p i p e - l i k e n e t w o r k o f l a t i c i f e r s r u n n i n g b e n e a t h t h e b a r k (3) may p r o v e t o be t h e b e s t e x p e r i m e n t a l m a t e r i a l f o r s t u d y i n g the biochemistry of rubber b i o s y n t h e s i s ; a species such as £ . argentatum t h a t produces rubber i n i n d i v i d u a l parenchyma c e l l s ( 6 ) , may p r o v i d e t h e s i m p l e s t g e n e t i c system. r

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B i o s y n t h e s i s o f Natural Rubber N a t u r a l r u b b e r i s p r o d u c e d by t h e a c t i o n o f t h e enzyme r u b b e r t r a n s f e r a s e (RuT) (EC 2 . 5 . 1 . 2 0 ) (6) on t h e s u b s t r a t e s i s o p e n t e n y l p y r o p h o s p h a t e ( I P P ) and a l l y l i c pyrophosphates, i n a side-branch of the ubiquitous i s o p r e n o i d pathway ( F i g u r e 1 ) . RuT, a p r e n y l t r a n s f e r a s e , r e q u i r e s d i v a l e n t c a t i o n s ( i n c l u d i n g Mg or Mn ) f o r a c t i v i t y , b u t RuT, I P P ( t h e s u b s t r a t e f o r 2+

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F i g u r e 1. The i s o p r e n o i d p a t h w a y , i l l u s t r a t i n g t h e p o s i t i o n of n a t u r a l rubber b i o s y n t h e s i s .

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e l o n g a t i o n ) and M g t o g e t h e r w i l l n o t r e s u l t i n r u b b e r f o r m a t i o n : a s e c o n d s u b s t r a t e , an a l l y l i c p y r o p h o s p h a t e , i s needed t o i n i t i a t e t h e p o l y m e r i z a t i o n p r o c e s s . A number o f a l l y l i c p y r o p h o s p h a t e s a r e c a p a b l e o f i n i t i a t i n g r u b b e r f o r m a t i o n i n i s o l a t e d washed r u b b e r particles (5). I t h a s been r e p o r t e d t h a t t h e e f f i c i e n c y of i n i t i a t i o n i n c r e a s e s w i t h the c h a i n l e n g t h of the i n i t i a t o r s (C < C < C < C ; 5 , 1 7 ) , although i t i s u n c l e a r how t h i s e f f e c t i s m e d i a t e d . The e n z y m a t i c s t e p s most i n t i m a t e l y c o n c e r n e d w i t h r u b b e r b i o s y n t h e s i s a r e shown i n F i g u r e 2 , and e n t a i l t h e i s o m e r i z a t i o n o f I P P t o d i m e t h y l a l l y l p y r o p h o s p h a t e (DMAPP), and t h e s y n t h e s i s o f C , C and C a l l y l i c p y r o p h o s p h a t e s ( i n a t r a n s c o n f i g u r a t i o n ) from I P P ( n o n - a l l y l i c C ) and DMAPP ( a l l y l i c C ) , b e f o r e RuT c a n form ç _ i s _ - l , 4 - p o l y i s o p r e n e from I P P . Hence, t h r e e b i o c h e m i c a l s t a g e s are i n v o l v e d i n r u b b e r m o l e c u l e f o r m a t i o n : (1) i n i t i a t i o n , w h i c h r e q u i r e s an a l l y l i c p y r o p h o s p h a t e s t a r t e r m o l e c u l e ; (2) elongation, w h i c h i s t h e p o l y m e r i z a t i o n i n t o r u b b e r o f i s o p r e n e u n i t s from i s o p e n t e n y l p y r o p h o s p h a t e ( I P P ) ; and (3) t e r m i n a t i o n , t h e r e l e a s e o f t h e c h a i n from t h e RuT enzyme, and w h i c h may o r may n o t be a d e d i c a t e d e n z y m a t i c process. 5

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For commercial success, production of high-grade n a t u r a l r u b b e r r e q u i r e s a s y s t e m t h a t p r o d u c e s many l o n g c h a i n rubber molecules as, g e n e r a l l y speaking, the longer the polyisoprene c h a i n the b e t t e r the high-performance c h a r a c t e r i s t i c s of the rubber. Nevertheless, short-chain r u b b e r p r o d u c e r s , s u c h as F i c u s e l a s t i c a p r o v i d e e x c e l l e n t s y s t e m s f o r i n v e s t i g a t i n g c h a i n t e r m i n a t i o n as t h e y have a much h i g h e r r a t i o o f t e r m i n a t i o n e v e n t s t o e l o n g a t i o n e v e n t s t h a n do l o n g - c h a i n p r o d u c e r s l i k e fl. b r a s i l i e n s i s or £ . argentatum. The b i o c h e m i s t r y o f t h e t e r m i n a t i o n mechanism has n o t b e e n t h o r o u g h l y i n v e s t i g a t e d , a l t h o u g h i t has b e e n shown t h a t t h e t e r m i n a l p y r o p h o s p h a t e c a n be h y d r o l y z e d , e s t e r i f i e d o r c y c l i z e d d e p e n d i n g on t h e s p e c i e s and c o n d i t i o n s ( T a n a k a , 1989). The i s o l a t i o n o f RuT and i t s t r a n s f e r and e x p r e s s i o n i n o t h e r systems i s p r o b a b l y i n s u f f i c i e n t i n i t s e l f t o a c h i e v e a new r u b b e r s u p p l y . Among many p o s s i b i l i t i e s , o t h e r p a r t s o f t h e i s o p r e n o i d pathway ( F i g u r e 1) may need t o be e x a m i n e d i n o r d e r t o o p t i m i z e t h e p r o d u c t i o n o f endogenous s u b s t r a t e s f o r r u b b e r p r o d u c t i o n i n m i c r o o r g a n i s m a l and p l a n t b i o s y n t h e t i c s y s t e m s , and a l s o t o



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prevent detrimental substrate d e f i c i e n c i e s further along the i s o p r e n o i d pathway. For example, the isoprene monomers a r e d e r i v e d f r o m i s o p e n t e n y l p y r o p h o s p h a t e ( I P P ) , w h i c h i s s y n t h e s i z e d from c a r b o h y d r a t e s v i a a c e t y l - C o A , HMG-CoA and m e v a l o n a t e ( F i g u r e 1 ) . The p r o d u c t i o n o f m e v a l o n a t e by t h e enzyme HMG-CoA r e d u c t a s e may be a r a t e - l i m i t i n g s t e p i n H - b r a s i l i e n s i s r u b b e r production (13). C o n s i d e r a b l e p r o g r e s s has a l r e a d y b e e n made as t h e fl. b r a s i l i e n s i s f a r n e s y l p y r o p h o s p h a t e ( F P P ) s y n t h e t a s e (12) and t h e F P P - s y n t h e t a s e (2) and I P P i s o m e r a s e (1) f r o m y e a s t have a l r e a d y been c h a r a c t e r i z e d .

L o c a l i z a t i o n o f Rubber T r a n s f e r a s e Rubber i s c o m p a r t m e n t a l i z e d i n t o c y t o p l a s m i c r u b b e r p a r t i c l e s b o t h i n l a t i c i f e r o u s s p e c i e s s u c h as H b r a s i l i e n s i s ( 3 ) , and i n s p e c i e s t h a t p r o d u c e r u b b e r i n parenchyma c e l l s s u c h as i n t h e b a r k o f P . a r g e n t a t u m (6). These two s p e c i e s a r e u n u s u a l among r u b b e r p r o d u c e r s as t h e y b o t h c a n make c o m m e r c i a l - g r a d e r u b b e r . During rubber b i o s y n t h e s i s , isopentenyl p y r o p h o s p h a t e i s o b t a i n e d from t h e aqueous e n v i r o n m e n t o u t s i d e t h e r u b b e r p a r t i c l e s and i s d e p h o s p h o r y l a t e d and p o l y m e r i z e d by t h e RuT enzyme; t h e d e v e l o p i n g i s o p r e n e polymers extend i n t o the p a r t i c l e i n t e r i o r . The f o r m a t i o n o f r u b b e r c a n be a s s a y e d by f o l l o w i n g t h e i n c o r p o r a t i o n o f l a b e l l e d i s o p r e n e from C - I P P i n t o newly-synthesized chains. I n o r d e r t o i s o l a t e t h e RuT enzyme i t i s f i r s t n e c e s s a r y t o d e t e r m i n e t h e l o c a t i o n o f the enzymatic r e a c t i o n . The enzymes r e s p o n s i b l e f o r r u b b e r b i o s y n t h e s i s may o r may n o t be a t t a c h e d t o t h e particles. Rubber p a r t i c l e s o f H . b r a s i l i e n s i s and P . argentatum i s o l a t e d by u s i n g a c e n t r i f u g a t i o n / f l o t a t i o n p r o c e d u r e ( 1 0 ) , had h i g h r u b b e r b i o s y n t h e t i c a c t i v i t y , a s a s s a y e d by t h e i n c o r p o r a t i o n o f r a d i o l a b e l f r o m C - I P P i n t o r u b b e r w i t h FPP as t h e i n i t i a t o r m o l e c u l e . H o w e v e r , no i s o p r e n e was i n c o r p o r a t e d i n t h e a b s e n c e o f a l l y l i c pyrophosphates. I n common w i t h o t h e r i s o p r e n o i d e n z y m e s , the r e a c t i o n r e q u i r e s d i v a l e n t c a t i o n s , p r o b a b l y Mg i n vivo. Repeated washing o f £ . argentatum rubber p a r t i c l e s , using the f l o t a t i o n / c e n t r i f u g a t i o n procedure, d i d n o t r e d u c e t h e l e v e l o f t h e h i g h l y a c t i v e bound r u b b e r t r a n s f e r a s e when a s s a y e d i n t h e p r e s e n c e o f I P P ( e l o n g a t i o n s u b s t r a t e ) and FPP ( i n i t i a t i o n s u b s t r a t e ) (10). S i m i l a r e x p e r i m e n t s have d e m o n s t r a t e d t h a t m o s t , i f n o t a l l , o f t h e RuT a c t i v i t y i n H . b r a s i l i e n s i s w h o l e l a t e x ( l a t e x t a p p e d f r o m l a t i c i f e r s and f r o m w h i c h no c o n s t i t u e n t s h a v e been removed) c a n a l s o be a c c o u n t e d f o r by an enzyme f i r m l y a t t a c h e d t o t h e washed r u b b e r p a r t i c l e s (WRPs; p u r i f i e d p a r t i c l e s from w h i c h t h e o t h e r c o n s t i t u e n t s o f w h o l e l a t e x have b e e n removed) ( C o r n i s h , K . , USDA-ARS, u n p u b l i s h e d r e s u l t s ) . E x p r e s s e d as a f u n c t i o n o f t h e number o f r u b b e r p a r t i c l e s a s s a y e d , no 1 4

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r e d u c t i o n o f RuT a c t i v i t y w i t h i n c r e a s i n g p u r i f i c a t i o n o f r u b b e r p a r t i c l e s was o b s e r v e d f o r e i t h e r s p e c i e s . Thus, t h e RuT m o l e c u l e s a r e f i r m l y a s s o c i a t e d w i t h r u b b e r p a r t i c l e s i n b o t h ft. b r a s i l i e n s i s ( 4 , 9 ) and £ . a r g e n t a t u m (8,10). However, L i g h t and D e n n i s (12) d e t e r m i n e d t h a t t h e c y t o s o l i c f r a c t i o n o f H . b r a s i l i e n s i s w h o l e l a t e x was e s s e n t i a l f o r r u b b e r b i o s y n t h e s i s b y S e p h a c r y l S-300 g e l f i l t e r e d p a r t i c l e s when a s s a y e d i n t h e p r e s e n c e o f C IPP. T h i s l e d them t o b e l i e v e t h a t , i n s t e a d o f a p a r t i c l e - b o u n d enzyme, a s o l u b l e RuT was p r e s e n t i n t h i s laticiferous species. ( I t i s n o t p o s s i b l e t o do t h e c o m p a r a b l e e x p e r i m e n t w i t h £ . a r g e n t a t u m as t h e p a r t i c l e s a r e p r e p a r e d f r o m a b a r k homogenate, n o t an i n t a c t latex.) T h e i r c o n c l u s i o n (12) was c o n s i s t e n t w i t h e a r l y work where a s o l u b l e p r o t e i n e x t r a c t from fi. b r a s i l i e n s i s l a t e x was t h o u g h t t o c a t a l y z e t h e e x t e n s i o n o f p r e e x i s t i n g r u b b e r p y r o p h o s p h a t e c h a i n s i n WRPs (4) and a s o l u b l e " r u b b e r t r a n s f e r a s e " was r e p o r t e d b y M c M u l l e n and McSweeney ( 1 5 ) . N e v e r t h e l e s s , none o f t h e s e s o l u b l e e x t r a c t s c o u l d s y n t h e s i z e rubber i n the absence of rubber particles. A l s o , i n t h e p r e s e n c e o f WRP, t h e y c o u l d be r e p l a c e d by a s e r i e s o f a l l y l i c p y r o p h o s p h a t e i n i t i a t o r molecules (5,9,14). Hence, i t i s l i k e l y t h a t t h e s e p r o t e i n s form p a r t o f t h e i n i t i a t i n g s y s t e m , s y n t h e s i z i n g a l l y l i c p y r o p h o s p h a t e s e s s e n t i a l t o t h e i n i t i a t i o n o f new rubber molecules, r a t h e r than p o l y i s o p r e n e formation itself. L i g h t and D e n n i s (12) went on t o i s o l a t e a s o l u b l e p r e n y l t r a n s f e r a s e ( F T ; monomer, 38 kD) as t h e a c t i v e component f r o m t h e l a t e x c y t o s o l . They f o u n d t h a t t h i s enzyme b e h a v e d , i n s o l u t i o n , a s an F P P - s y n t h e t a s e , as I have a l s o c o n f i r m e d ( C o r n i s h , K . , USDA-ARS, u n p u b l i s h e d r e s u l t s ; p u r i f i e d PT was a g e n e r o u s g i f t f r o m Genentech). N e v e r t h e l e s s , i n the presence of g e l f i l t e r e d p a r t i c l e s , I P P , DTT and M g t h i s PT i n d u c e d t h e production of rubber. I n s i m i l a r e x p e r i m e n t s , WRP f r o m H - b r a s i l i e n s i s l a t e x a s s a y e d i n t h e p r e s e n c e o f I P P and M g had a much l o w e r r a t e o f I P P - i n c o r p o r a t i o n t h a n w h o l e l a t e x ( C o r n i s h , Κ . , USDA-ARS, u n p u b l i s h e d r e s u l t s ) . Whole l a t e x a c t i v i t y c o u l d be p a r t i a l l y r e s t o r e d t o t h e WRP by t h e a d d i t i o n o f P T . H o w e v e r , when t h e WRP were a s s a y e d i n t h e p r e s e n c e o f FPP as w e l l a s I P P , t h e bound RuT s u p p o r t e d much h i g h e r r a t e s o f I P P - i n c o r p o r a t i o n t h a n w h o l e l a t e x , o r WRP p l u s p r e n y l t r a n s f e r a s e .


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2+

2+

The Role o f P r e n y l T r a n s f e r a s e i n Rubber B i o s y n t h e s i s An a l t e r n a t i v e e x p l a n a t i o n t o a c c o u n t f o r t h e o b s e r v e d s t i m u l a t o r y e f f e c t o f t h e p r e n y l t r a n s f e r a s e on r u b b e r b i o s y n t h e s i s , o t h e r t h a n i t a c t u a l l y b e i n g R u T , was s t i l l l e f t open. C o u l d PT be s u p p l y i n g t h e s o u r c e o f a l l y l i c p y r o p h o s p h a t e n e c e s s a r y t o i n i t i a t e de_ novo r u b b e r biosynthesis? T h i s seems l i k e l y b e c a u s e , a s d i s c u s s e d e a r l i e r , PT b e h a v e s as an F P P - s y n t h e t a s e i n t h e a b s e n c e



24


o f r u b b e r p a r t i c l e s ( 1 2 ) , and FPP i s a p o t e n t i n i t i a t o r (5,9,10,14). I f PT i s s t i m u l a t i n g r u b b e r f o r m a t i o n by s y n t h e s i z i n g FPP i n i t i a t o r m o l e c u l e s , t h e n t h i s p r e s u p p o s e s t h e p r e s e n c e o f e i t h e r DMAPP o r I P P - i s o m e r a s e i n the i n v i t r o assay (see F i g u r e 2 ) : p r e n y l t r a n s f e r a s e s c a n n o t make a l l y l i c p y r o p h o s p h a t e s i n t h e a b s e n c e o f DMAPP. Using a s p e c i f i c i n h i b i t o r of the isomerase ( 3 , 4 oxido-3-methyl-l-butyl diphosphate; 16), k i n d l y supplied by C . D a l e P o u l t e r ( U n i v e r s i t y o f U t a h ) , c o n s i d e r a b l e I P P - i s o m e r a s e a c t i v i t y was d e t e c t e d i n w h o l e l a t e x and a l s o a l o w l e v e l o f c o n t a m i n a t i o n i n t h e PT p r e p a r a t i o n i t s e l f ( C o r n i s h , Κ . , USDA-ARS, u n p u b l i s h e d r e s u l t s ) . As o n l y one i n i t i a t o r m o l e c u l e i s r e q u i r e d f o r e a c h r u b b e r m o l e c u l e , made f r o m p e r h a p s t h o u s a n d s o f i s o p r e n e m o l e c u l e s , a v e r y s m a l l amount o f DMAPP o r I P P - i s o m e r a s e i n a rubber p a r t i c l e p r e p a r a t i o n would permit the s y n t h e s i s o f enough FPP t o a l l o w a c o n s i d e r a b l e production of rubber. Of c o u r s e , t h e d e m o n s t r a t i o n o f a r o l e o f t h e L i g h t and D e n n i s (12) PT i n r u b b e r m o l e c u l e i n i t i a t i o n d o e s n o t r u l e o u t an a d d i t i o n a l r o l e i n r u b b e r e l o n g a t i o n . E x a m i n i n g t h e e f f e c t o f PT i n t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f FPP ( t h e s u b s t a n c e made by PT i n s o l u t i o n ) s h o u l d r e v e a l any a d d i t i o n a l r o l e . PT h a d no e f f e c t on I P P - i n c o r p o r a t i o n by w h o l e l a t e x e v e n a t s a t u r a t i n g FPP c o n c e n t r a t i o n s ( C o r n i s h , Κ . , USDA-ARS, unpublished r e s u l t s ) . The p o s s i b i l i t y t h a t t h i s n e g a t i v e r e s u l t was c a u s e d by a s a t u r a t i n g l e v e l o f PT a l r e a d y p r e s e n t i n w h o l e l a t e x was d i s c o u n t e d as t h e [ F P P ] dependence o f I P P - i n c o r p o r a t i o n by w h o l e l a t e x was i d e n t i c a l t o t h a t o f WRP ( f r o m w h i c h t h e s o l u b l e components o f w h o l e l a t e x , i n c l u d i n g P T , have been removed) ( C o r n i s h , Κ . , USDA-ARS, u n p u b l i s h e d r e s u l t s ) . P a r t i c l e - B o u n d Rubber T r a n s f e r a s e The k i n e t i c s o f I P P - i n c o r p o r a t i o n by £ . a r g e n t a t u m WRP (10) a r e s t r a i g h t - f o r w a r d c o n c e n t r a t i o n d e p e n d e n c i e s on b o t h I P P and F P P . B o t h s u b s t r a t e s g i v e r i s e t o l i n e a r E a d i e - H o f s t e e p l o t s w h i c h a r e i n d i c a t i v e o f s i n g l e enzyme systems ( 1 0 ) . S i m i l a r experiments using H . b r a s i l i e n s i s WRP d e m o n s t r a t e d t h a t t h i s RuT b e h a v e s , b i o c h e m i c a l l y , i n a manner v i r t u a l l y i d e n t i c a l t o t h e £ . a r g e n t a t u m R u T . The a p p a r e n t K , f o r FPP r a n g e s b e t w e e n 0 . 8 and 3 uM, f o r IPP between 250 and 500 uM, and f o r t h e c o f a c t o r Mg between 70 and 80 uM. These b i o c h e m i c a l s t u d i e s s u g g e s t t h a t s i n g l e enzymes a r e r e s p o n s i b l e f o r r u b b e r m o l e c u l e i n i t i a t i o n by FPP and p o l y m e r i z a t i o n o f I P P . A l s o , i t seems p r o b a b l e t h a t t h e two p r o c e s s e s a r e c a t a l y z e d by a s i n g l e enzyme a l t h o u g h an enzyme c o m p l e x h a s y e t t o be ruled out. I f a complex does e x i s t , the a c t i v e s i t e s r e s p o n s i b l e f o r t h e two p r o c e s s e s must be s p a t i a l l y adjacent. 2+



3. CORNISH

Natural Rubber

Biosynthesis

25

P r o t e i n a n a l y s i s o f WRP has been a t t e m p t e d i n e f f o r t s t o d i s t i n g u i s h t h e t r u e RuT enzyme f r o m o t h e r particle-bound proteins. Despite the biochemical s i m i l a r i t y o f t h e H . b r a s i l i e n s i s and £ . a r g e n t a t u m r u b b e r b i o s y n t h e t i c s y s t e m s , t h e WRP p r o t e i n s p r o v e d quite distinct. S i l v e r - s t a i n i n g o f o n e - d i m e n s i o n a l SDSPAGE g e l s r e v e a l e d t h a t fl. b r a s i l i e n s i s WRP p o s s e s s a t l e a s t 17 d i f f e r e n t p r o t e i n s ( C o r n i s h , Κ . , USDA-ARS, u n p u b l i s h e d r e s u l t s ) w h e r e a s £ . a r g e n t a t u m WRP have o n l y 4-8 ( 7 ) . The £ . a r g e n t a t u m 4 8 . 5 kD g l y c o p r o t e i n , w h i c h i s t h e most a b u n d a n t r u b b e r p a r t i c l e p r o t e i n i n t h i s s p e c i e s (7) may be RuT i t s e l f ( 8 ) . T h i s p r o t e i n has been c h a r a c t e r i z e d (7) a n d , a t t h i s t i m e , most o f i t s gene h a s been i s o l a t e d ( B a c k h a u s , R . A . , A r i z o n a S t a t e U n i v e r s i t y , personal communication, 1991). Experiments i n d i c a t e t h a t t h i s p r o t e i n i s l o c a t e d l a r g e l y w i t h i n the rubber p a r t i c l e , b u t a t t h e s u r f a c e w i t h t h e g l y c o s y l a t e d end protruding i n t o the cytoplasm (7). T h i s i s an a p p r o p r i a t e l o c a t i o n f o r RuT, w h i c h must p o l y m e r i z e a hydrophobic polymer to the p a r t i c l e i n t e r i o r w h i l e o b t a i n i n g h y d r o p h i l i c s u b s t r a t e s from the c y t o s o l . The most a b u n d a n t p a r t i c l e - b o u n d p r o t e i n i n Η · b r a s i l i e n s i s r u b b e r p a r t i c l e s i s t h e 1 4 . 6 kD " r u b b e r elongation factor" polypeptide (11). T h i s 1 4 . 6 kD p o l y p e p t i d e may p l a y a r o l e i n r u b b e r b i o s y n t h e s i s a l t h o u g h t h i s h a s n o t been c o n f i r m e d . Nevertheless, p o l y c l o n a l a n t i b o d i e s r a i s e d a g a i n s t i t d i d , i n one e x p e r i m e n t , i n h i b i t RuT a c t i v i t y i n w h o l e l a t e x ( 1 1 ) . Conclusions R u b b e r p a r t i c l e s i s o l a t e d from fî. b r a s i l i e n s i s and £ . a r g e n t a t u m s y n t h e s i z e new r u b b e r when i n c u b a t e d w i t h I P P , FPP and M g . The a c t i v i t y o f RuT i s n o t d i m i n i s h e d b y r e p e a t e d w a s h i n g , d e m o n s t r a t i n g t h a t t h e enzyme i s f i r m l y bound t o t h e r u b b e r p a r t i c l e i n b o t h s p e c i e s . Also, b i o c h e m i c a l s t u d i e s o f r u b b e r b i o s y n t h e s i s by i s o l a t e d r u b b e r p a r t i c l e s o f fl. b r a s i l i e n s i s and £ . a r g e n t a t u m s u g g e s t t h a t t h e two b i o s y n t h e t i c s y s t e m s a r e e x t r e m e l y similar. E v i d e n c e s u g g e s t s t h a t t h e £ . a r g e n t a t u m RuT may be t h e a b u n d a n t 4 8 . 5 kD p a r t i c l e - b o u n d g l y c o p r o t e i n . W h i c h o f t h e Η· b r a s i l i e n s i s p a r t i c l e - b o u n d p r o t e i n s i s RuT has y e t t o be d e t e r m i n e d . A c o n s i d e r a b l e mass o f i n f o r m a t i o n has been a c c u m u l a t e d on t h e b i o l o g i c a l mechanism o f r u b b e r biosynthesis. T h i s has i n c l u d e d t h e i s o l a t i o n and c l o n i n g o f g e n e s f o r enzymes i n v o l v e d i n t h e p r o d u c t i o n o f a l l y l i c p y r o p h o s p h a t e i n i t i a t o r s f o r new r u b b e r molecule. The d e f i n i t i v e i s o l a t i o n o f t h e r u b b e r t r a n s f e r a s e enzyme r e s p o n s i b l e f o r r u b b e r m o l e c u l e e l o n g a t i o n , and t h e n i t s g e n e , i s t h e f i n a l s t e p r e q u i r e d b e f o r e t r a n s f o r m a t i o n e x p e r i m e n t s on p o t e n t i a l d o m e s t i c r u b b e r - p r o d u c i n g s p e c i e s c a n be r e a l i s t i c a l l y b e g u n . 2+



26


References 1. Anderson, M.S.; Muehlbacher, M.; Proffitt, J.; Poulter, C.D. J . Biol. Chem. 1989, 264, 19169 2. Anderson, M.S., Yarger, J.G.; Burck, C.L.; Poulter, C.D. J . Biol. Chem. 1989, 264, 191 3. d'Auzac, J.; Jacob, J.L.; Chrestin, H. Physiology of Rubber Tree Latex, CRC Press, Inc. 1989. 4. Archer, B.L.; Audley, B.G.; Cockbain, E.G.; McSweeney, G.P. Biochem. J . 1963, 89, 565 5. Archer, B.L.; Audley, B.G. Bot. J . Linn. Soc. 1987, 94, 181 6. Backhaus, R.A. Israel J . Botany 1985, 34, 283 7. Backhaus, R.A.; Cornish, K.; Chen, S.F.; Huang, D.S.; Bess, V.H. Phytochem 1991, 30, 2493 8. Benedict, C.R.; S. Madhavan, S.; Greenblatt, G.A.; Venkatachalam, K.V.; Foster, M.A. Plant Physiol. 1990, 92, 816 9. Berndt, J . 1963, U.S. Government Res. Rep. AD-601: 729 10. Cornish, K.; Backhaus, R.A. Phytochem. 1990, 29, 3809 11. Dennis, M.S.; Light, D.A. J . Biol. Chem. 1989, 264, 18608 12. Light, D.R.; Dennis, M.S. J.Biol. Chem. 1989, 264, 18589 13. Lynen, F. J . Rubber Res. Inst. Malays. 1969, 21, 389 14. Madhavan, S.; Greenblatt, G.A.; Foster, M.A.; Benedict, C.R. Plant Physiol. 1989, 89, 506 15. McMullen, A. J.; McSweeney, G.P. Biochem. J . 1966, 101, 42 16. Muehlbacher, M.; Poulter, C.D. Biochemistry 1988, 27, 7315 17. Tanaka, Y. Prog. Polym. Sci. 1989, 14, 339 RECEIVED

December 26, 1991


Regulation of Isopentenoid Metabolism - American Chemical Society

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