Biotechnology of Amylodextrin Oligosaccharides - American Chemical

Chapter 4

Substrate-Based Investigations of the Active Site of CGTase Enzymatic Syntheses of Regioselectively Modified Cyclodextrins

Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch004

Sylvain Cottaz and Hugues Driguez

Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, B.P. 53 X, 38041 Grenoble cedex, France

New i n s i g h t s have been obtained on the s p e c i f i c i t y of the catalytic site of CGTase of Bacillus macerans by enzymatic conversion of modified m a l t o s y l f l u o r i d e s . The c a t a l y t i c c a p a b i l i t y of this enzyme led to the first regioselective synthesis of modified c y c l o d e x t r i n i n good y i e l d . Furthermore, under coupling conditions, new acceptors l e d to r e a c t i o n products suggesting at l e a s t four s u b s i t e s f o r the c a t a l y t i c s i t e of t h i s enzyme.

Cyclodextrin glycosyltransferase (EC 2.4.1.19) i s an enzyme which c a t a l y z e s the r e v e r s i b l e t r a n s f e r r e a c t i o n of g l u c o s y l u n i t s between maltodextrins : disproportionation G

n

+

G

m

«

*

G

+

n-y

G

m+y

cyclization G

n

« * coupling

C D

x

+

G

(n-x)

0097-6156/91/0458-0044$06.00/0 © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


4.

COTTAZ & DRIGUEZ

45

Active Site of CGTase

The cyclisation reaction of linear maltoo l i g o s a c c h a r i d e s i n t o α , β and γ - c y c l o d e x t r i n s (CD) and the c o u p l i n g r e a c t i o n which leads to modified l i n e a r dextrins on the reducing end are special disproportionation reactions. The binding of an o l i g o s a c c h a r i d e to a protein involves a number of hydrogen bonds, hydrophobic interactions a n d V a n d e r Waals c o n t a c t s between the s u g a r and amino a c i d r e s i d u e s . This area of contact defines the g l o b a l b i n d i n g s i t e . It is convenient to e x p r e s s t h e b i n d i n g o f an o l i g o s a c c h a r i d e as t h e sum o f t h e i n t e r a c t i o n s between e a c h m o n o s a c c h a r i d e u n i t and t h e c o r r e s p o n d i n g b i n d i n g s i t e . These i n d i v i d u a l b i n d i n g sites or subsites prove very useful for studying s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s or f o r the l o c a l i z a t i o n of the catalytic site (usually situated between two subsites). In o r d e r t o o b t a i n i n f o r m a t i o n a b o u t t h e number o f s u b s i t e s i n t h e c a t a l y t i c s i t e o f CGTase f r o m Klebsiella pneumoniae, Bender (1) studied the cyclization and disproportionation reactions with various maltooligos a c c h a r i d e s f r o m DP 2-8 and a m a l t o d e x t r i n DP 1 9 . S i n c e m a l t o o c t a o s e p r o v e d t o be t h e s m a l l e s t oligosaccharide f o r d i r e c t c y c l i z a t i o n and t h a t DP 19 i s n o t a b e t t e r s u b s t r a t e , i t was s u g g e s t e d t h a t t h e a c t i v e s i t e o f t h i s enzyme c o n s i s t s o f 8 s u b s i t e s . To d e f i n e t h e s p e c i f i c i t y o f t h i s a c t i v e s i t e o n l y t h e c o u p l i n g r e a c t i o n was s t u d i e d by u s i n g m o d i f i e d CDS (2,3) o r m o d i f i e d a c c e p t o r s ( 4 - 6 ) . T h e s e a p p r o a c h e s have shown t h a t a b u l k y or charged groups on C-6 o f a glucosyl unit prevent this modified glucosyl residue f r o m f i t t i n g i n t o s u b s i t e s S and R o f t h e d o n o r p a r t o f the active site (Figure 1) . These studies also demonstrated that subsites Τ and U may accomodate various modified glucosyl acceptors ; for instance, alkyl and aryl aor β-D-glucosides, methyl a-Dxyloside, methyl 6-0-methyl-a-D glucoside, panose, s u c r o s e and m a l t o b i u r o n i c a c i d a r e a c c e p t o r s . The a i m o f t h i s work i s t o p r e s e n t a new a p p r o a c h based on the autocondensation reaction of modified maltosyl fluorides for the determination of the specificity of the active site, and on t h e coupling r e a c t i o n on 1 , 6 - a n h y d r o - m a l t o o l i g o s a c c h a r i d e s t o o b t a i n i n f o r m a t i o n on t h e number o f subsites.

MATERIALS and EXPERIMENTAL Enzymes CGTase was a gift of Amano Co s u b t i l i s i n was o b t a i n e d f r o m S i g m a .

Ltd.

(Japan)


and

46

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES

Chemicals The d e t a i l s o f t h e p r e p a r a t i o n and c h a r a c t e r i z a t i o n o f m a l t o s y l f l u o r i d e s 1-10 w i l l be r e p o r t e d e l s e w h e r e . The corresponding acetylated derivatives gave elemental a n a l y s i s a n d s p e c t r a l d a t a ( C nmr a n d MS) i n a c c o r d with the proposed structure. Catalytic deacetylation w i t h sodium methoxide i n m e t h a n o l was p e r f o r m e d just before incubations. The p u r i t y of the deacetylated compounds was c h e c k e d by H . P . L . C . (column : NH μBondapack 10 |im ; e l u e n t : a c e t o n i t r i l e - w a t e r 7:3). 6 -O-Acetyl-a-maltosyl fluoride 10 was obtained from α - m a l t o s y l f l u o r i d e 11 (7) by t r a n s a c e t y l a t i o n of v i n y l acetate i n p y r i d i n e i n the presence of s u b t i l i s i n . 1 , 6 - A n h y d r o - m a l t o s e 13 and 1,6-anhydro-maltotriose 14 were s y n t h e s i z e d as a l r e a d y d e s c r i b e d ( 8 , 9 ) . 1 3

2


1

Enzymatic

Incubation with

CGTase

Coupling Reactions for the S p e c i f i c i t y of Acceptor Part of the A c t i v e S i t e . - Various potential substrates (5 mg) a n d CDS (5 mg) were i n c u b a t e d w i t h CGTase (635 U / m l ; 20 μ ΐ ) i n p h o s p h a t e b u f f e r (0.1 M, pH 7, 1 ml) f o r 1 h a t 4 5 C . The m i x t u r e was t h e n a n a l y z e d by H . P . L . C . as already described. e

S p e c i f i c i t y o f t h e Donor P a r t o f C G T a s e . - F l u o r i d e s 111 ( 50 mM) i n p h o s p h a t e b u f f e r were i n c u b a t e d w i t h CGTase (20 μ ΐ / m l ) f o r 12 h a t 4 5 C , and t h e n t h e m i x t u r e was a n a l y z e d b y H . P . L . C . e

RESULTS and DISCUSSION The r e s u l t s o f e x p e r i m e n t s w h e r e i n α - C D and CGTase were i n c u b a t e d w i t h a l l t h e f l u o r i d e s 1-11 a r e shown on t h e r i g h t p a r t o f t a b l e 1. I n e a c h c a s e , H . P . L . C . analysis of the mixture showed that a l l - the compounds are a c c e p t o r s . These r e s u l t s c o n f i r m the poor s p e c i f i c i t y o f t h e Τ and U s u b s i t e s . To a p p r e c i a t e t h e s p e c i f i c i t y o f R and S s u b s i t e s , the f l u o r i d e s 1-11 were i n c u b a t e d w i t h CGTase b u t i n the a b s e n c e o f α - C D . A l l t h e m o d i f i c a t i o n s made a t t h e 6 p o s i t i o n of the reducing u n i t prevent b i n d i n g i n subsite S ( l e f t p a r t o f t a b l e 1 ) . The o n l y m o d i f i c a t i o n s a l l o w e d are the acetylation and t h e methylation of primary hydroxyl group of the non reducing unit ; the 4thiomaltosyl fluoride 9 is also recognized. By preparative H.P.L.C. and characterization by mass s p e c t r o m e t r y , u s i n g t h e FAB t e c h n i q u e , i t h a s b e e n shown that alternating oligosaccharides are formed, w i t h the s t a r t i n g m o d i f i e d d i s a c c h a r i d e as t h e r e p e a t i n g u n i t . In two c a s e s , f o r 8 and 10, c y c l i c compounds 15 and 16



4.

COTTAZ & DRIGUEZ


47

'Table I . S t r u c t u r e o f the m o d i f i e d m a l t o s y l f l u o r i d e s 1-11 and t h e i r e n z y m a t i c b e h a v i o u r under c o u p l i n g and c y c l i z a t i o n c o n d i tions

COMPOUND X X ζ

1

2

3

4

5

6

7

8

9

10

11

Η

F

Br

OMc

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

H

F

Br

OMc

OH

OAc

OH

0

0

0

0

0

0

0

0

S

0

0

American Chemical Society Library 1155 16th St., N.W. In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; Washington, U.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. 1 3

A

c

E

F i g u r e 2. 75 MHz O n . m . r . spectrum o f 6 , 6 , 6 - t r i - ( 9 - m e t h y l c y c l o m a l t o h e x a o s e i n D2O u s i n g DEPT sequence. Primed numbers r e f e r t o the carbons o f the methylated s u b s t r a t e s .


4. COTTAZ & DRIGUEZ


49 E

were a l s o i s o l a t e d . T h e s e compounds a r e β , 6 , 6 - t r i - 0 s u b s t i t u t e d a-CDS. C - N . m . r . s p e c t r a o f t h e s e compounds show a simple pattern compatible with a regular disaccharide repeating unit and a l s o match t h e Co molecular symmetry (fig. 2 ) . 6 ,6 ,6 -Tri-0-methyl c y c l o m a l t o h e x a o s e was o b t a i n e d i n an o v e r a l l y i e l d o f c a . 12% from t h e c o m m e r c i a l l y a v a i l a b l e maltose (10). C

1 3


A

C

E

The incubation of modified maltosyl fluorides confirmed some features already reported in the literature, b u t a l s o b r i n g some new i n f o r m a t i o n a b o u t t h e s p e c i f i c i t y o f t h e a c t i v e s i t e o f C G T a s e . The amino a c i d s o f s u b s i t e s Τ a n d U do n o t e s t a b l i s h essential b o n d i n g s w i t h p r i m a r y h y d r o x y l s o f m a l t o s y l r e s i d u e s . On the donor p a r t , the subsite S does n o t a c c e p t any m o d i f i c a t i o n of these p o s i t i o n s , but R i s l e s s s p e c i f i c . The same s p e c i f i c i t y was a l s o f o u n d i n TaJca-amylase (11) and was confirmed when linear dextrins from the condensation of 6 -O-methyl-maltosyl fluoride were incubated with Ta/ca-amylase to afford 6 -0-methylmaltose. To t r y t o d e t e r m i n e t h e number o f s u b s i t e s o f t h e acceptor part of the active site we i n c u b a t e d 1,6a n h y d r o - m a l t o s e 13 a n d m a l t o t r i o s e 14 w i t h CGTase i n t h e presence of α - C D . Only compound 14 gave coupling p r o d u c t s a t a r e a s o n a b l e r a t e . I t i s s u g g e s t e d t h a t two g l u c o s y l r e s i d u e s a r e r e c o g n i z e d on a c o u p l i n g r e a c t i o n so o n l y two s u b s i t e s c o n s t i t u t e t h e a c c e p t o r p a r t o f t h e active site. Reactions are i n progress f o r the determination of t h e number o f s u b s i t e s on t h e d o n o r p a r t o f t h e a c t i v e site. 1

!

Literature Cited 1.

2. 3. 4.

5. 6. 7.

Bender, H. "Proc. 4th Intern. Symposium on Cyclodextrins". In Advances in Inclusion Science ; Huber, O., Szejtli, J., Eds ; Kluver Academic Publishers: Dordrecht, 1988 ; p 19. Kobayashi, S. ; Lee Ashraf, W.R. ; Braun, P. ; French, D. Starch/Stärke 1988, 40, 112-16. Nagamine, Y. ; Sumikawa, M. ; Omichi, K. ; Ikenaka, T. J. Biochem. 1987, 102, 767-75. French, D. ; Levine, M.L. ; Norberg, E. ; Nordin, P. ; Pazur, J.H. ; Wild, G.M. J. Am. Chem. Soc. 1954, 76, 2387-90. Kitahata, S. ; Okada, S. ; Fukui, T. Agric. Biol. Chem. 1978, 42, 2369-74. Wheeler, M. ; Hanke, P. ; Weill, E. Arch. Biochem. Biophys. 1963, 102, 397-9. Hehre, E.J. ; Mizokami, K. ; Kitahata, S. Denpun Kagaku 1983, 30, 76.


50

BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES

8.

Fujimaki, I. ; Ichikawa, Y. ; Kuzuhara, H. Carbohydr. Res. 1982, 101, 148-51. 9. Sakairi, N. ; Hayashida, M. ; Kuzuhara, H. Carbohydr. Res. 1989, 185, 91-104. 10. Cottaz, S. ; Driguez, H., J. Chem. Soc., Chem. Commun. 1989, 16, 1088-9. 11. Arita, H. ; Isemura, M., Ikenaka, T., Matsushima, Y. Bull. Chem. Soc. Jpn. 1970, 43, 818-23.


RECEIVED October 3, 1990


Biotechnology of Amylodextrin Oligosaccharides - American Chemical

Recommend Documents