Crystal Structure of Biliverdin Reductase A heme (Fe-porphyrin complex) is a prosthetic g r o u p o f h e m o p r o t e i n s s u c h a s h e m o g l o b i n , my og lo bi n, cy to ch ro me s an d so on . In bi ol og ic al systems, the heme is decomposed by two enzymes ( h e m e o x y g e n a s e a n d b i l i v e r d i n r e d u c t a s e ) a n d e x c r e t e d ( F i g . 1 ) . B i l i v e r d i n r e d u c t a s e ( B V R ) catalyzes the last step of the heme catabolism, in which the biliverdin is reduced into bilirubin with two e l e c t r o n s f r o m N A D ( P ) H . R e d u c t i o n o f b i l i v e r d i n (formation of bilirubin) is important for the disposal of the heme catabolite formed in the fetus since the p l a c e n t a i s p e r m e a b l e t o b i l i r u b i n b u t n o t t o b i l i v e r d i n . Bi li ru bi n is th e mo st ab un da nt bi ol og ic al an ti -o xi da nt i n m a m m a l i a n t i s s u e s , a n d i s h i g h l y r e l a t e d t o neuroprotection, b u t a l s o t o c y t o t o x i c i t y ( k e r n i c t e r u s ) . C l e a r l y , B V R i s a k e y e n z y m e a n d l o g i c a l pharmacological target for controlling bilirubin level in situ . We hav e ana lyz ed the cry sta l str uct ure of rat BVR at 1.4 Å resolution ( Fig. 2 ), whose diffraction data was colle cted at beaml ine BL44B 2 [1]. This e n z y m e c o n s i s ts o f tw o d o m a i n s th a t a r e p a c k e d tightly together. The N-terminal domain (123 amino ac id s) is a ch ar ac te ri st ic of a di nu cl eo ti de bi nd in g f o l d , t h e s o - c a l l e d R o s s m a n n f o l d , w h i l e t h e C - te rm in al do ma in (1 69 am in o ac id s) co nt ai ns si x β - s t r a n d s a n d e i g h t h e l i c e s , a n d i s d o m i n a t e d b y a l a r g e , a n d p r e d o m i n a n t l y a n t i p a r a l l e l s i x - s t r a n d e sheet. NAD(P)H Binding to BVR : The Rossmann fold in the N-terminal domain is the most likely NAD(P)H bin din g sit e. The ‘fi nge rpr int ’ reg ion ( Gly 15- Val 16- Gly17-Arg18-Ala19-Gly20 ) and a ‘hydrophobic core’ ( Va l1 1, Va l1 3, Le u2 4, Le u2 7, Va l4 2 ) ar e fo un d in this region. Glu96 is apparently capable to interact with the nicotinamide ring of NAD(P)H through the hydrogen bond ( Fig. 3 ). Indeed, Glu96Ala mutant of BVR, in which Glu96 was replaced with Ala, did n o t e x h i b i t t h e e n z y m a t i c a c t i v i t y . A u n i q u e property of BVR is its ability to use either NADH or NADPH at different pH optima; NADH is used in the l o w e r p H r a n g e o f 6 . 7 - 6 . 9 , w h e r e a s N A D P H i s used at the higher pH of 8.7 [2]. In a model study, i n w h i c h N A D H o r N A D P H w a s p u t o n t h e Rossmann fold of BVR, we found that Arg44-Arg45- Glu46 is located very close to the 2’-position of the adenosine ribose ( Fig. 3 ). In combination with the m u t a t i o n a l s t u d i e s , w e p r o p o s e d t h a t A r g 4 4 a n d Ar g4 5 pl ay cr uc ia l ro le s in NA DP H bi nd in g, wh il e Glu46 modulates the NADH binding. B i l i v e r d i n B i n d i n g t o B V R : T h e p u t a t i v e N A D ( P ) H b i n d i n g s i t e i s o n t h e l o w e r s i d e o f t h e p o c k e t t h a t i s c o n s t r u c t e d b e t w e e n t h e N - a n d C - t e r m i n a l d o m a i n s . O n t h e o t h e r h a n d , t h e r e a r e l o c a t e d f o u r b a s i c r e s i d u e s , A r g 1 7 1 , L y s 2 1 8 , A r g 2 2 4 a n d A r g 2 2 6 , o n t h e u p p e r s i d e o f t h i s pocket ( Fig. 3 ). Since the biliverdin recognition by BVR has been proposed to be achieved through an Fig. 1. Heme degradation pathway in mammals. 9 bilirubin-IX α biliverdin-IX α heme Heme Oxygenaze glucuronosyl transferase Biliverdine-IX α Reductase excretion glucuronic acid bilirubin-IX α biliverdin-IX α heme References [1] A. Kikuchi, S.-Y. Park, H. Miyatake, D. Sun, M. Sato, T. Yoshida and Y. Shiro, Nature Strl. Biol. 8 (2001) 221. [2] R. K. Kutty and M. D. Maines, J. Biol. Chem. 256 (2981) 3956. [ 3 ] O . C u n n i n g h a m e t a l . , J . B i o l . C h e m . 2 7 5 (2000) 19009. e l e c t r o s t a t i c i n t e r a c t i o n b e t w e e n t h e n e g a t i v e l y c h a r g e d p r o p i o n a t e s i d e c h a i n s o f b i l i v e r d i n a n d p o s i t i v e l y c h a r g e d r e s i d u e s o f B V R [ 3 ] , w e proposed that cluster of the four basic r e s i d u e s i s a p o s s i b l e b i l i v e r d i n b i n d i n g s i t e . T h e p o c k e t i s w i d e e n o u g h t o a c c o m m o d a t e b o t h b i l i v e r d i n a n d N A D ( P ) H . W h e n b i l i v e r d i n w o u l d b i n d t o t h i s s i t e , t h e d i s t a n c e b e t w e e n t h e r e d u c t i o n s i t e ( C 1 0 m e s o posit ion) of biliv erdin and t h e N A D ( P ) H n i c o t i n a m i d e is estimated to be ~1 0 Å. In th is mo de l, Tyr 97 is lo ca te d be tw ee n th e ni co ti na mi de and C10 meso position, and therefore this r e s i d u e m a y m e d i a t e a h y d r i d e ( H – ) transfer from NAD(P)H to biliverdin in the enzymatic reaction. Fig. 2. Stereo view of the overall structure of rat BVR. Akihiro Kikuchi and Yoshitsugu Shiro SPring-8 / RIKEN E-mail: yshiro @ postman.riken.go.jp F i g . 3 . T h e p r o p o s e d b i n d i n g s i t e s f o r N A D ( P ) H (electron donor) and substrate (biliverdin) in rat BVR. C C N N 10 ‘fingerprint’ region R44 R45 E46 E96 Y97 R171 R224 R226 K218 E46 R44 R45 ‘fingerprint’ region E96 Y97 R226 R224 R171 K218
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