X a n t h i n e o x i d o r e d u c t a s e c a t a l y z e s t h e h y d r o x y l a t i o n o f h y p o x a n t h i n e a n d x a n t h i n e , t h e l a s t t w o s t e p s i n t h e f o r m a t i o n o f u r a t e i n p u r i n e catabolism in man. The enzyme is a target of drugs not onl y aga ins t gou t [1] , but als o aga ins t oxy gen radical-induced tissue damage [2]. Allopurinol has been used as a widely used drug effective against g o u t a n d h y p e r u r i c e m i a s i n c e i t w a s i n t r o d u c e d m o r e t h a n 3 0 y e a r s a g o . B o v i n e m i l k x a n t h i n e o x i d a s e i s a n a r c h e t y p a l e n z y m e , w h i c h w a s originally described as aldehyde oxidase in 1902 [3] a n d h a s b e e n t h e b e n c h m a r k o f t h e m e t a l l o - fla vop rot ein s [4, 5]. The enz yme fro m mam mal ian s o u r c e s , i n c l u d i n g m a n , i s s y n t h e s i z e d a s t h e dehydrogenase form but it can be readily converted t o t h e o x i d a s e f o r m b y o x i d a t i o n o f s u l f h y d r y l r e s i d u e s o r b y p r o t e o l y s i s . T h e d e h y d r o g e n a s e shows a prefe rence for NAD + reduc tion, while the o x i d a s e f a i l s t o r e a c t w i t h N A D + a n d e x c l u s i v e l y u s e s o x y g e n m o l e c u l e a s i t s s u b s t r a t e l e a d i n g t o t h e f o r m a t i o n o f s u p e r o x i d e a n i o n a n d h y d r o g e n High-Resolution Structure of Bovine Milk Xanthine Oxidoreductase and Inhibitor Complexes peroxide ( Fig. 1 ). The enzyme has been implicated in di se as es ch ar ac te ri ze d by ox yg en ra di ca l- in du ce d ti ss ue da ma ge , su ch as po st is ch em ic re pe rf us io n i n j u r y [ 2 ] . T h e c r y s t a l s t r u c t u r e s o f x a n t h i n e o x i d o r e d u c t a s e i n t h e t w o f o r m s , d e h y d r o g e n a s e a n d o x i d a s e , h a v e b e e n s o l v e d a f t e r s u c c e s s f u l crystallization of both forms of the enzyme, to clarify the structure-based mechanism of conversion [6,7]. The experiment was car out at beamline BL40B2 . The active form of the enzyme is a homodimer o f m o l e c u l a r m a s s 2 9 0 k D a , w i t h e a c h o f t h e monomers acting independently in catalysis. Each subunit molecule is composed of an N-terminal 20 k D a d o m a i n c o n t a i n i n g t w o i r o n s u l f u r c e n t e r s , a ce nt ra l 40 kD a FA D do ma in , an d a C- te rm in al 85 k D a m o l y b d o p t e r i n - b i n d i n g d o m a i n w i t h t h e f o u r r e d o x c e n t e r s a l i g n e d i n a n a l m o s t l i n e a r f a s h i o n ( Fig. 2 ). The hydroxylation of xanthine takes place a t t h e m o l y b d o p t e r i n c e n t e r ( M o - p t ) a n d t h e electrons thus introduced are rapidly transferred to t h e o t h e r l i n e a r l y a l i g n e d r e d o x c e n t e r s a s illustrated in Fig. 1 . The reaction catalysed by the M o h y d r o x y l a s e s i s d i s t i n c t f r o m t h o s e o f o t h e r bi ol og ic al hy dr ox yl at io n sy st em s li ke P4 50 in th at a n o x y g e n a t o m i s i n c o r p o r a t e d i n t o t h e p r o d u c t from water rather than from an O 2 molecule [5] . In HN N H N H N O O Xanthine Uric Acid HN N H H N O O O N H Mo(VI) -Pterin Fe/S I Fe/S II FAD monomer: 1332 amino acid residues Inhibitors (allopurinol, TEI-6720 ) H 2 O Mo( IV ) -Pterin NAD + NADH O 2 H 2 O 2 + O 2 − − Dehydrogenase Oxidase F i g . 1 . R e a c t i o n s c h e m e o f x a n t h i n e d e h y d r o g e n a s e a n d x a n t h i n e o x i d a s e . T h e enzyme is a dimer ( MW 290,000) having two identical subunits of 1332 amino acids. 11 (B) t h e c r y s t a l s t r u c t u r e o f t h e a c t i v e e n z y m e , w e assigned a double-bonded sulfur atom, a double- bonded oxygen atom (=O), and an oxygen atom with a single bond (OH) as ligands to the Mo ion. It should be noted that the protein environments of t w o o x y g e n l i g a n d s a r e d i s t i n c t i n t h e h i g h re so lu ti on cr ys ta l st ru ct ur e an d th e OH li ga nd is c o n s i d e r e d t o b e l a b i l e o x y g e n b a s e d o n t h e structure of the alloxanthine enzyme complex (to be published). In addition to the crystal structure of th e co mp le x of th e en zy me an d al lo xa nt hi ne , the oxidative product of allopurinol, we solved the c r y s t a l s t r u c t u r e o f t h e c o m p l e x o f t h e e n z y m e and TEI-6720, a strong candidate for a new anti- gout drug. In the crystal structure of the enzyme T E I - 6 7 2 0 c o m p l e x , n u m e r o u s h y d r o g e n b o n d s a n d h y d r o p h o b i c i n t e r a c t i o n s w e r e o b s e r v e d b e t w e e n t h e p r o t e i n a n d t h i s e x t r e m e l y p o t e n t i n h i b i t o r ( F i g . 3 ) , a n d s o m e o f t h e m s e e m e d t o c o n t r i b u t e t o s t r o n g b i n d i n g i n a s i m i l a r w a y t o substrate recognition [8]. The FAD active site is the part of the enzyme t h a t s h o w s t h e l a r g e s t d i f f e r e n c e b e t w e e n x a n t h i n e d e h y d r o g e n a s e a n d o x i d a s e [ 1 , 2 ] . C l e a v a g e o f a s u r f a c e - e x p o s e d l o o p o f X D H cau ses maj or str uct ura l rea rra nge men t of a loo p cl os e to th e fl av in ri ng ( Fi g. 4 ). Th is mo ve me nt partially blocks NAD substrate access to the FAD c o f a c t o r a n d c h a n g e s t h e e l e c t r o s t a t i c environment of the active site, accounting for the s w i t c h o f s u b s t r a t e s p e c i f i c i t y o b s e r v e d f o r t h e conversion between the two forms. F i g . 2 . ( A ) C r y s t a l s t r u c t u r e o f t h e x a n t h i n e d e h y d r o g e n a s e d i m e r d i v i d e d i n t o t h e t h r e e major domains. The enzyme contains salicylate, a competitive inhibitor. From N- to C-terminus, t h e d o m a i n s a r e : i r o n - s u l f u r - c e n t e r d o m a i n ( r e s i d u e s 1 - 1 6 5 ; r e d ) , F A D d o m a i n ( r e s i d u e s 226 - 531; gree n), and Mo-p t doma in (res idue s 590 - 1332; blue). Cofactors are also included. ( B ) T h e a r r a n g e m e n t o f t h e c o f a c t o r s a n d s a l i c y l a t e i n o n e s u b u n i t o f t h e e n z y m e a r e presented. The Mo ion is in green, the iron ions are in light blue, and the sulfur atoms in yellow. (A) Lys569 Leu219 Cys992 FAD Mo-pt a b 7.8Å 5.0Å 12.3Å Salicylate Fe/S II Fe/S I 5.9Å 12 Thr1010 Arg880 Glu802 Asn768 3.11 2.84 2.79 2.67 2.83 References [1] G.B. Elion, Science 244 (1989) 41. [2] J.M. McCord, N. Engl. J. Med. 312 (1985) 159. [ 3 ] F . S c h a r d i n g e r , Z . U n t e r s u c h . N a h r u n g s Genussmittel 5 (1902) 1113. [4] T. Nishino, J. Biochem. (Tokyo) 11 6 ( 19 94 ) 1. [5] R. Hille and T. Nishino, FASEB J. 9 (1995) 995. [6] B.T. Eger et al. , Acta Cryst. D 56 (2000) 1656. [ 7 ] C . E n r o t h e t a l . , P r o c . N a t l . A c a d . S c i . U S A (2000) 10723. [8] K. Okamoto et al ., in preparation. Fig. 4. Ribbon diagram of the FAD and Fe/S II active sites. The active site loop from Asp 426 to Asp 434 wh ic h ch an ge s it s co nf or ma ti on du ri ng th e XD H to XO transition is shown in light blue for XDH and in green for XO. The positions of residues Asp 429 and A r g 4 2 6 a r e i n d i c a t e d ; t h e i r s i d e c h a i n s s h o w dramatic shifts in position and are major contributors to the change in electrostatic charge at the flavin site. Fig. 3. Active site structure at the molybdenum center of TEI-6720 inhibitor bound form. The molybdenum atom is shown in cyan. T a k e s h i N i s h i n o a , K e n O k a m o t o a a n d Emil F. Pai b (a) Nippon Medical School (b) The University of Toronto E-mail: nishino @ nms.ac.jp Thr262 FAD Phe337 Glu45 Asp429* Arg426* Asp429 Arg426 Gly46 Fe/S II a b Thr1010 Arg880 Glu802 Asn768 3.11Å 2.84Å 2.79Å 2.67Å 2.83Å 13
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