Chapter 42
C e l l u l a s e s o f Cellulomonas fimi
The Enzymes and Their Interactions with Substrate D. G. Kilburn, N. R. Gilkes, R. C. Miller, Jr., and R. A. J. Warren Department of Microbiology, University of British Columbia, 300—6174 University Boulevard, Vancouver, British Columbia V 6 T 1W5, Canada
An exoglucanase and an endoglucanase of the bacterium Cellulomonasfimiare glycoproteins which bind strongly to microcrystalline cellulose. Each protein comprises two functionally independent domains joined by a sequence of proline and threonine residues: a catalytic domain which does not bind to cellulose; and a cellulose-binding domain which is not enzymatically active. The cellulose-binding domain is at the N-terminus of the endoglucanase but at the C-terminus of the exoglucanase. A C. fimi protease cleaves both enzymes to release the independently functioning domains. The glycosyl groups on the proteins protect them from cleavage by the protease when they are bound to cellulose. M i c r o o r g a n i s m s use several types o f e x t r a c e l l u l a r enzymes t o degrade cellulose t o glucose: exoglucanases ( 1 , 4 - / ? - D - g l u c a n cellobiohydrolases, E . C . 3 . 2 . 9 1 ) ; endoglucanases (endo- 1,4-/?-D-glucan glucanohydrolases, E . C . 3.2.1.4); a n d , d e p e n d i n g o n the o r g a n i s m , cellobiases (/?-D-glucoside g l u c o hydrolases, E . C . 3 . 2 . 1 . 2 1 ) . I n recent years, these enzymes have received considerable a t t e n t i o n because o f their possible use i n the conversion o f waste b i o m a s s , such as s t r a w , sawdust a n d bagasse, t o useful c h e m i c a l s . A n o u t c o m e o f t h i s w o r k has been the r e a l i z a t i o n t h a t cellulases are o f great interest i n themselves, irrespective o f their c o m m e r c i a l p o t e n t i a l (1). A given m i c r o o r g a n i s m m a y p r o d u c e one or more enzymes o f each t y p e . A n u n d e r s t a n d i n g o f the role o f each e n z y m e i n cellulose biodégradation requires their p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n , a n d a n a n a l y s i s o f t h e ways i n w h i c h they interact w i t h the substrate a n d w i t h each other. H o w e v e r , i t is often quite difficult t o determine the n u m b e r a n d t y p e o f t r u l y different enzymes p r o d u c e d b y a n o r g a n i s m . M a n y c e l l u l o l y t i c m i c r o o r g a n i s m s secrete proteases, w h i c h m a y degrade some or a l l o f the cellulases t o s m a l l e r , 0097-6156/89/0399-0587$06.00/0 © 1989 American Chemical Society
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PLANT C E L L W A L L P O L Y M E R S
a c t i v e species. F u r t h e r m o r e , cellulases f r o m b o t h p r o c a r y o t i c a n d e u c a r y o t i c m i c r o o r g a n i s m s m a y be g l y c o s y l a t e d , a n d d e g l y c o s y l a t i o n in vivo c a n give rise to a p p a r e n t l y different forms of a n e n z y m e . A w i d e l y used a p p r o a c h to resolve such p r o b l e m s is the c l o n i n g of the s t r u c t u r a l genes for cellulases (2). T h e n u c l e o t i d e sequence of a gene c a n be used to predict the a m i n o a c i d sequence of the cellulase i t encodes, w h i c h i n t u r n can be used to m a k e p r e d i c t i o n s about the s t r u c t u r e of the cellulase. A l l of t h i s i n f o r m a t i o n can be used to make comparisons between cellulases f r o m a single o r g a n i s m a n d f r o m different o r g a n i s m s . E x p r e s s i o n of a cloned gene i n a n a p p r o p r i a t e host gives i n t a c t e n z y m e u n c o n t a m i n a t e d w i t h other cellulases. If the n a t i v e e n z y m e is g l y c o s y l a t e d , expression of its gene i n Eschericha coli gives the n o n - g l y c o s y l a t e d f o r m of the e n z y m e . C o m p a r i s o n of t h i s w i t h the i n t a c t , n a t i v e e n z y m e w i l l reveal the effects of g l y c o s y l a t i o n . C e l l u l a s e s o f Cellulomonas
fimi
W h e n g r o w n o n cellulosic substrates, the b a c t e r i u m Cellulomonas fimi (3) p r o d u c e s a c o m p l e x a r r a y o f cellulases, some o f w h i c h are g l y c o s y l a t e d (4-6). Its cellulase profile varies w i t h b o t h the n a t u r e of the s u b s t r a t e a n d w i t h c u l t u r e age, p o s s i b l y as a consequence of proteolysis a n d degl y c o s y l a t i o n (6). A n exoglucanase ( C e x ) a n d a n endoglucanase ( C e n A ) b i n d to the substrate i n cultures g r o w n w i t h A v i c e l , a m i c r o c r y s t a l l i n e cellulose, a n d they can be recovered i n t a c t f r o m the r e s i d u a l A v i c e l i n s u c h cultures (6). T h i s f a c i l i t a t e d their p u r i f i c a t i o n t o homogeneity b y subsequent f a s t - p r o t e i n - l i q u i d - c h r o m a t o g r a p h y (7). B o t h were g l y c o p r o teins (6). B o t h enzymes h y d r o l y z e d c a r b o x y m e t h y l c e l l u l o s e ( C M C ) , a l t h o u g h w i t h different k i n e t i c s (8); b o t h released r e d u c i n g sugar f r o m A v i cel; b u t o n l y C e x h y d r o l y z e d p - n i t r o p h e n y l c e l l o b i o s i d e ( p N P C ) a n d 4m e t h y l u m b e l l i f e r y l c e l l o b i o s i d e ( M U C ) . B o t h proteins were m o n o m e r s of very s i m i l a r size: C e x contained 443 a n d C e n A 418 a m i n o acids. E a c h p r o t e i n was c o m p o s e d o f three discrete segments: a sequence o f 20 a m i n o acids composed of o n l y p r o l y l a n d t h r e o n y l residues, t e r m e d the P r o - T h r b o x , w h i c h was almost perfectly conserved; a sequence of about 100 a m i n o acids w h i c h was r i c h i n h y d r o x y a m i n o acids, of low charge density, a n d 5 0 % conserved; a n d a sequence of about 300 a m i n o acids w h i c h h a d a r e l a t i v e l y h i g h charge density, b u t was not conserved (9). T h e order of the segments was reversed i n the two enzymes ( F i g . 1). C o m p a r i s o n o f t h e N a t i v e a n d R e c o m b i n a n t F o r m s o f C. fimi C e l lulases I n s t r u c t u r a l t e r m s , the o n l y difference between n a t i v e C e x a n d C e n A a n d the r e c o m b i n a n t forms of the enzymes p r o d u c e d i n E. coli was t h a t the former were g l y c o s y l a t e d . F o r s i m p l i c i t y , the g l y c o s y l a t e d f o r m s are referred to as g C e x a n d g C e n A , a n d the n o n - g l y c o s y l a t e d forms as n g C e x and ngCenA. G l y c o s y l a t i o n d i d not affect the s u b s t r a t e specificities of C e x a n d C e n A ; it h a d very l i t t l e effect o n their c a t a l y t i c a c t i v i t i e s ; a n d i t d i d not
N
Ν
1 2 PT
Cex
CenA CHARGED
LOW CHARGE HYDROXYL RICH
316 335
PT
418
443
COOH
COOH
F i g u r e 1. O v e r a l l s t r u c t u r e s o f a n exoglucanase ( C e x ) a n d a n endoglucanase ( C e n A ) f r o m C. fimi. P T denotes a P r o - T h r b o x ; t h e n u m b e r s refer t o a m i n o a c i d residues.
CHARGED
112 134
LOW CHARGE HYDROXYL RICH
590
PLANT C E L L W A L L P O L Y M E R S
affect t h e i r s t a b i l i t i e s to heat a n d p H (10). B o t h f o r m s o f the enzymes b o u n d to A v i c e l , w h i c h f a c i l i t a t e d t h e i r p u r i f i c a t i o n f r o m E. coli ( 1 1 , 1 2 ) . C. fimi secreted a serine protease w h i c h was a c t i v e against the c e l l u lases. I n s o l u t i o n , the g l y c o s y l a t e d forms o f C e x a n d C e n A were cleaved m u c h m o r e s l o w l y t h a n the n o n - g l y c o s y l a t e d forms, a n d the cleavage sites a p p e a r e d different i n the t w o forms ( N . R . G i l k e s , u n p u b l i s h e d observat i o n s ) . However, w h e n the enzymes were b o u n d to A v i c e l , the g l y c o s y l a t e d forms of C e x a n d C e n A were resistant to the protease, b u t the n o n g l y c o s y l a t e d forms r e m a i n e d sensitive (10) ( F i g . 2). T h e r e f o r e , one f u n c t i o n of g l y c o s y l a t i o n was to protect C e x a n d C e n A against p r o t e o l y s i s , e s p e c i a l l y w h e n b o u n d to cellulose. I n t e r a c t i o n s o f C. fimi C e l l u l a s e s w i t h C e l l u l o s e Cleavage of A v i c e l - b o u n d n g C e x a n d n g C e n A released c a t a l y t i c a l l y a c t i v e fragments f r o m the A v i c e l ( F i g . 2), suggesting t h a t C e x a n d C e n A were organized into two independently functioning domains, a substrate-binding d o m a i n a n d a c a t a l y t i c d o m a i n . T h i s was confirmed b y a n a l y s i s o f the cleavage p r o d u c t s released b y the a c t i o n of the protease o n n g C e x a n d n g C e n A i n s o l u t i o n (12). B o t h enzymes were degraded to discrete fragments ( F i g . 3). A n a l y s i s of the fragments showed t h a t the p r i m a r y cleavage of n g C e x gave fragments of M 30 k D a a n d 20 k D a ( F i g . 4). I n each case, the large f r a g m e n t r e t a i n e d c a t a l y t i c a c t i v i t y b u t d i d not b i n d t o A v i c e l , whereas the smaller f r a g m e n t was c a t a l y t i c a l l y i n a c t i v e b u t c o u l d b i n d to A v i c e l . T h e a c t u a l site of cleavage i n b o t h cases was at the c a r b o x y l t e r m i n u s of the P r o - T h r b o x ( F i g . 4.) It was quite clear t h a t i n C e x a n d C e n A , the 5 0 % conserved regions t h a t h a d low charge density, a n d were r i c h i n h y d r o x y a m i n o acids, were the s u b s t r a t e - b i n d i n g d o m a i n s . I n each e n z y m e , the b i n d i n g d o m a i n was separated f r o m the c a t a l y t i c d o m a i n b y a P r o - T h r b o x . Since the P r o - T h r b o x is q u i t e s i m i l a r to the hinge region o f I g A i i m m u n o g l o b u l i n s (13), i t is t e m p t i n g to speculate t h a t the P r o - T h r b o x f u n c t i o n s as a hinge also, thereby a l l o w i n g the c a t a l y t i c d o m a i n to m o v e over the surface of a cellulose fibril i n spite of the e n z y m e b e i n g anchored at one e n d . R e t e n t i o n of t h e i r i n d i v i d u a l properties w h e n separated b y proteolysis showed q u i t e c l e a r l y t h a t the two d o m a i n s f u n c t i o n e d i n d e p e n d e n t l y . T h i s was e m p h a s i z e d for the c a t a l y t i c d o m a i n s b y the properties of a fusion p o l y p e p t i d e i n w h i c h the c a t a l y t i c d o m a i n , P r o - T h r b o x , a n d the first 32 a m i n o acids of the s u b s t r a t e b i n d i n g d o m a i n of C e x were fused to most of the c a t a l y t i c d o m a i n o f C e n A ( F i g . 5). T h e fusion p o l y p e p t i d e h a d b o t h exoglucanase a n d endoglucanase a c t i v i t y (14). r
A l t h o u g h its two d o m a i n s c o u l d f u n c t i o n i n d e p e n d e n t l y , r e m o v a l of the s u b s t r a t e - b i n d i n g d o m a i n of n g C e n A reduced e n z y m a t i c a c t i v i t y against m i c r o c r y s t a l l i n e cellulose b u t not against C M C or a m o r p h o u s cellulose (12). T h i s suggested t h a t the s u b s t r a t e - b i n d i n g d o m a i n p l a y e d a c r i t i c a l role i n the h y d r o l y s i s of c r y s t a l l i n e cellulose. T h e gene, cenB, for a second endoglucanase, C e n B , of C. fimi, was also cloned i n E. coli ( 1 1 , 1 5 ) . T h e p o l y p e p t i d e expressed f r o m cenB i n
42.
KILBURN ETA L
Celluloses of Cellulomonas fimi
591
F i g u r e 2. Z y m o g r a m of g C e n A ( A - I ) a n d n g C e n A ( J - Q ) after i n c u b a t i o n w i t h C. fimi protease. C e l l u l a s e s , b o u n d to A v i c e l , were i n c u b a t e d w i t h protease or c o n t r o l buffer for 72 hr at 30° C , t h e n centrifuged to give cellulose-bound ( A - E , J - N ) a n d s u p e r n a t a n t ( F - I , O - Q ) f r a c t i o n s . P r o d u c t s were separated o n a S D S gel, r e p l i c a t e d onto C M C - a g a r o s e a n d developed w i t h C o n g o r e d . A , J . buffer c o n t r o l ( 4 ° C i n c u b a t i o n ) ; B , F , K , 0 , protease; C , G , L , P , protease + P M S F c o n t r o l ; D , H , M , Q , buffer c o n t r o l ; Ε,Ι,Ν, buffer + P M S F control.
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PLANT C E L L W A L L P O L Y M E R S
F i g u r e 3. T i m e course of proteolysis of n g C e n A a n d n g C e x . 300 μg of n g C e n A ( A ) or n g C e x ( B ) dissolved i n 725 μΐ of phosphate buffer c o n t a i n i n g 1.5 u n i t s of crude C. fimi protease were i n c u b a t e d at 3 7 ° C . R e a c t i o n s were s a m p l e d at 0, 6, 24, 48, 96, a n d 144 h (lanes 1-6, r e s p e c t i v e l y ) , treated w i t h P M S F , a n d a n a l y z e d by S D S - P A G E ( 8 % a c r y l a m i d e ) . C o n t r o l samples were i n c u b a t e d i n the absence of protease for 144 h (lane 7). A l l lanes were l o a d e d w i t h sample equivalent to 2.8 /zg of i n i t i a l p r o t e i n .
F i g u r e 4. S c h e m a t i c representation of cleavage of n g C e n A a n d n g C e x b y C. fimi protease. T h e p r i m a r y cleavage sites (solid triangles) were defined b y a m i n o - t e r m i n a l sequence analyses, W e s t e r n b l o t analyses, a n d the a p p a r e n t m o l e c u l a r masses of a p p r o p r i a t e fragments, i n r e l a t i o n t o the d e d u c e d a m i n o a c i d sequences of n g C e n A a n d n g C e x . T h e p r o t e i n s are d r a w n a p p r o x i m a t e l y t o scale. S t i p p l e d areas represent c a t a l y t i c d o m a i n s : cross-hatched areas, cellulose b i n d i n g d o m a i n s ; u n s h a d e d areas, P r o - T h r b o x h i n g e regions. N u m b e r s refer t o a m i n o a c i d residues, b e g i n n i n g at the a m i n o t e r m i n i of the m a t u r e p r o t e i n s f r o m C. fimi.
H N 2
H N 2
2
Η Ν
CATALYTIC DOMAIN
} >
FUSION
COOH
CATALYTIC DOMAIN CenA
COOH
BINDING DOMAIN
F i g u r e 5. S c h e m a t i c of the s t r u c t u r e of the C e x - C e n A fusion p r o t e i n . s t i p p l e d areas are P r o - T h r boxes.
CATALYTIC DOMAIN Cex
BINDING DOMAIN
CenA
CATALYTIC DOMAIN
Cex
The
COOH
42.
KILBURN ET AL.
Celluloses of Cellulomonas fimi
595
E. coli had a M of 110 kDa. It also could be bound to Avicel, but its structural relatedness to Cex and CenA has not yet been determined. A deletion mutant of cenB encoded a polypeptide of M 70 kDa. The missing segment represented the carboxyl terminal 40 kDa or CenB. The 70 kDa fragment had enzymatic activity and it could still bind to substrate (11). The nature and function of the 40 kDa carboxyl terminus of CenB are being determined. r
r
Similarity of C. fimi Cellulases to Trichoderma reesei Cellulases Analysis of the genes for four cellulases of the basidiomycete T. reesei showed that these proteins had a bifunctional organization remarkably similar to that of Cex and CenA of C. fimi, and again with reversal of domain order in pairs of the four enzymes (16). The T. reesei enzymes could also be cleaved into separate domains by proteolysis, and this was discussed elsewhere (Claeyssens, M., and Tomme, P., this volume). Suffice it to emphasize here that the genes encoding cellulases in C. fimi and T. reesei appear to have arisen by domain shuffling and that the enzymes they encode appear to interact with cellulose in a comparable manner, i.e., a catalytic domain is held on the substrate by a binding domain. Cellulases from the bacterium Clostridium thermocellum also contained sequences analogous to Pro-Thr boxes as well as highly conserved carboxyl terminal sequences (2,17,18). It remains to be seen if they have functional organizations similar to those of the C fimi and T. reesei enzymes. Acknowledgment We thank the Natural Sciences and Engineering Research Council of Canada for financial support. Literature Cited 1. Aubert, J.-P.; Beguin, P.; Millet, J . , Eds. Biochemistry and Genetics of Cellulose Degradation; FEMS Symp. No. 43; Academic Press: San Diego, 1988. 2. Beguin, P.; Gilkes, N. R.; Kilburn, D. G.; Miller, R. C., Jr.; O'Neill, G. P.; Warren, R. A. J . CRC Crit. Rev. Biotechnol. 1987, 6, 129. 3. Stackenbrandt, E.; Kandler, O. Int. J. Syst. Bacteriol. 1979, 29, 273. 4. Beguin, P.; Eisen, H.; Roupas, A. J. Gen. Microbiol. 1977, 101, 191. 5. Beguin, P.; Eisen, H. Eur. J. Biochem. 1978, 87, 525. 6. Langsford, M . L.; Gilkes, N. R.; Wakarchuk, W. W.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J . J. Gen. Microbiol. 1984, 130, 1367. 7. Langsford, M . L. Ph.D. Thesis, University of British Columbia, 1988. 8. Gilkes, N. R.; Langsford, M . L.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J . J. Biol. Chem. 1984, 259, 10455. 9. Warren, R. A. J.; Beck, C. F.; Gilkes, N. R.; Kilburn, D. G.; Langsford, M. L.; Miller, R. C., Jr.; O'Neill, G. P.; Scheufens, M.; Wong, W. K. R. Proteins 1986, 1, 335.
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10. Langsford, M . L.; Gilkes, N. R.; Singh, B.; Moser, B.; Miller, R. C., Jr.; Warren, R. A. J.; Kilburn, D. G . FEBS Lett. 1987, 225, 163. 11. Owolabi, J.; Beguin, P.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J . Appl. Environ. Microbiol. 1988, 54, 518. 12. Gilkes, N. R.; Warren, R. A. J.; Miller, R. C., Jr.; Kilburn, D. G . J. Biol. Chem. 1988, 263, 10401. 13. Frangione, B.; Wolfenstein-Todel, C. Proc. Nat. Acad. Sci. U.S.A. 1972, 69, 3673. 14. Warren, R. A. J.; Gerhard, B.; Gilkes, N. R.; Owolabi, J . B.; Kilburn, D. G.; Miller, R. C., Jr. Gene 1987, 61, 421. 15. Gilkes, N. R.; Kilburn, D. G.; Langsford, M . L.; Miller, R. C . , Jr.; Wakarchuk, W. W.; Warren, R. A. J.; Whittle, D. J.; Wong, W. K. R. J. Gen. Microbiol. 1984, 130, 1377. 16. Knowles, J.; Lehtovaara, P.; Teeri, T . TrendsBiotechnol.1987, 5, 255. 17. Beguin, P.; Cornet, P.; Aubert, J.-P. J. Bacteriol 1985, 162, 102. 18. Grepinet, O.; Beguin, P. Nucleic Acids Res. 1986, 14, 1791. RECEIVED May 19, 1989
