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S t r u c tu r e , e v o l u ti o n a n d a c t io n o f v i ta m i n B s - d e p e n d e n t e n z y m e sJohan N JansoniusT h e n u m b e r o f k n o w n t h r e e - d i m e n s i o n a l s t ru c t u r e s o f v i ta m i nB 6 - d e p e n d e n t e n z y m e s h a s d o u b l e d i n t h e p a s t t w o y e a r s . Af o u r t h t y p e o f f o l d f o r B 6 - d e p e n d e n t e n z y m e s , i n v o l v i n g a T I M -b a r r e l d o m a i n , h a s b e e n d i s c o v e r e d . A l a n i n e r a c e m a s e is th ef i r s t k n o w n r e p r e s e n t a t i v e o f t h i s n e w f o l d . S i g n i f i c a n t p r o g r e s sh a s b e e n m a d e i n u n d e r s t a n d i n g t h e a l l o s t e r i c e f f e c t s i n t h et r y p t o p h a n s y n t h a s e r e a c t i o n .A d d r e s s e sBiozentrum, Univers ity o f Basel , K l inge lbergst rasse 70, CH -405 6,Basel , Switzer land; e-mai l : jansonius@ubaclu .un ibas.chC u r r e n t O p i n i o n i n S t r u c t u r a l B i o l o g y 1998 , 8 :759 -?69h t tp : / / b iomedne t . com/e lec re f /0959440X00800759 C ur ren t B io logy L td ISSN 0959 -440XA b b r e v i a t i o n sA - A aminoacry la teA A T a s p a r t a t e aminotransferaseA R a la n in e r a c e ma s eB C A T branched-chain amino acid aminotransferaseC B L c y s t a t h io n in e 13-1yaseD A A T o - a min o a c id aminotransferaseDGD d ia lky lg lyc ine decarboxy laseF-IPP 5-f luoro- indo le propanol phosphateG P g lycero l-3-phosphateG S A g l u t a m a t e - 1semia ldehydeG S A T G S A a min o mu t a s eIGP indo leg lycero lphosphateIPP indo le-3-propanol phosphateOA SS O-acety lser ine su l fhydry laseO A T ornithine aminotransferaseO D C o r n i th in e d e c a r b o x yla s ePDB Prote in Data BankP L P p y r id o xa l 5 ' - p h o s p h a t ePM P pyr idoxamine 5 ' -phosphatePPL-D-Ala phosphopyr idoxyl-o-a lan ineTIL t ryptophan indo le-lyaseTN threonine d e a m i n a s eTPL tyrosine phenol- lyaseTRPS t ryptophan synthaseTR PSI} TRP S 13 subun it(e -A P T (o -am ino c id :py ruva te am ino trans fe rase

I n t r o d u c t i o nP y r i d o x a l 5 ' - p h o s p h a t e ( P L P ) a n d p y r i d o x a m i n e 5 ' - p h o s -p h a t e ( P M P ) a r e t h e f o r m s o f v i t a m i n B 6 t h a t a c t a sc o f a c to r s fo r a m i n o a c id m e t a b o l i s i n g e n z y m e s . I n v i t a m i nB 6 - d e p e n d e n t e n z y m e s , P L P i s i n v a r i a b ly b o u n d t o a na c t iv e s i t e l y s in e th ro u g h a S c h i f f b a s e l i n k a g e , t h e ' i n t e r -n a l ' a l d i m i n e ( F i g u r e 1 , t h e ' e x t e r n a l ' a l d i m i n e i s m a d ew i t h t h e s u b s t r a t e a m i n o g r o u p ). I t s 5 ' - p h o s p h a t e g r o u p i ss e c u r e l y a t ta c h e d t o t h e p r o t e i n m a t r i x t h r o u g h u p t o n i n eh y d r o g e n b o n d s a n d , o f t e n , c h a r g e i n t e r a c t i o n s . F o r P M P ,t h e p h o s p h a t e g r o u p i s t h e m a i n a n ch o r . T h e e l e c t r o n s i n kp r o p e r t i e s o f p y r i d o xa l , t h e m o s t v e r s a t il e o r g a n i c c a t a ly s tk n o w n , a r e u t i l is e d i n o r d e r t o e n d o w t h e h o l o e n z y m e w i t hs u c h d i v e r s e r e a c t i o n s p e c i f i c i t i e s a s t r a n s a m i n a t i o n ,r a c e m is a t io n , o ~ d e c a rb o x y la t io n , a ld o l c l e a v a g e , a n d 13

a n d "f e l i m i n a t i o n a n d r e p l a c e m e n t r e a c ti o n s . T h r o u g h a ni n g e n i o u s i n t e r p l a y b e t w e e n c o f a c t o r a n d a c t i v e s i t e o r ga n -i s a ti o n , t h e r e q u i r e d r e a c t i o n i s o p t i m i s e d w h i l e a llp o s s i b l e o th e r s a r e n e a r ly c o m p l e t e l y p r e v e n t e d [ 1,2 ]. P L Pe n z y m e s t h u s h a v e e v o l v e d t o b e h i g h l y e f f i c i e n t i n t e r m so f s p e e d , a s w e l l as i n t e rm s o f r e a c t io n a n d s u b s t r a t e s p e c i -f i c i ty [3 ]. F ig u re 1 d e p ic t s s o m e o f t h e k e y c a t a ly t i cin t e rm e d ia t e s o c c u r r in g in th e r e a c t io n s l i s t e d a b o v e .T h e f i rs t t h r e e - d i m e n s i o n a l s t r u c t u r e o f a B 6 e n z y m e t o b ed e t e r m i n e d w a s , in 1 9 80 , t h a t o f a sp a r t a t e a m i n o t r a n s -f e r a s e ( A A T ) [ 4 ] , t h e m o s t t h o r o u g h l y i n v e s t i g a t e dB 6 e n z y m e [3, 5, 6] . I t t o o k e ig h t y e a r s b e fo re th e s e c o n ds t r u c t u r e o f a B 6 e n z y m e w a s p u b l i s h e d , t h a t o f t h e t r y p t o -p h a n s y n t h a s e O~z~ c o m p l e x ( T R P S ) [ 7] . S i n c e th e n , t h ei m p a c t o f m o d e r n f a s t d a ta c o l l e ct i o n m e t h o d s h a s g r e a tl ya c c e l e r a t e d t h e s t r u c t u r e d e t e r m i n a t i o n o f t h e s e r a t h e rl a rg e m o l e c u l e s , w h i c h h a v e m o l e c u l a r w e i g h t s t h a t a r era re ly u n d e r 9 0 , 0 0 0 . In th e n e x t e ig h t y e a r p e r io d , t h es t r u c t u r e s o f o - a m i n o a c i d : p y r u v a t e a m i n o t r a n s f e r a s e ( o~ -A P T ) [ 8] , d i a l k y l g l y c in e d e c a r b o x y l a s e ( D G D ) [ 9, 10 ],t y r o s i n e p h e n o l - l y a s e ( T P L ) [ 1 1 ] , o r n i t h i n e d e c a r b o x y l a s e(O D C ) [1 2, 13 ], c y s t a th io n in e ~ - ly a s e (C B L ) [1 4 ] a n dD - a m i n o a c i d a m i n o t r a n s f e r a s e ( D A A T ) [ 1 5 ] w e r e e l u c i -d a t e d . W i t h t w o e x c e p t i o n s , a l l t h e s e e n z y m e s a r e q u i t eo b v i o u s l y e v o l u t io n a r i l y r e l a t e d t o A A T . T h e y a r e a ll c o m -p a c t h o m o d i m e r s o r l a r g e r o l i g o m e r s w i t h d i h e d r a ls y m m e t r y . T h e i r s u b u n i t s h a v e , i n g e n e ra l , t w o o r t h r e ed o m a i n s . T h e c e n t r a l P L P - b i n d i n g d o m a i n i s a n o p e n o t / 1 3s t r u c tu r e , w i t h a u n i q u e s e v e n - s t r a n d e d ~ s h e e t o f t o p o l o -g y a g fe d b c , i n w h ic h a l l t h e s t r a n d s , e x c e p t g , a re p a ra l l e l .T h e a c t i v e s i t e l y s i n e r e s i d e s i n a l o o p b e t w e e n s t r a n d s fa n d g . B o t h s u b u n i t s o f t h e ' c a t a ly t i c d i m e r ' c o n t r i b u t e t oe a c h a c t iv e s i t e , w h ic h l i e s a t t h e s u b u n i t a n d d o m a inin te r f a c e (F ig u re 2 ) .T h e t w o u n r e l at e d e n z y m e s a r e T R P S a n d D A A T . T h et r y p t o p h a n s y n t h a s e ~ s u b u n i t ( T R P S ~ ) r e p r e s e n t s a s e c -o n d f o ld t y p e f o r a B 6 e n z y m e ( t h e ~ s u b u n i t i s t h ea b u n d a n t o r /l ] o r ' T I M ' - b a r r e l t y p e ) . D A A T a l s o h a s an o v e l f o ld . A l t h o u g h i t is a n i n t i m a t e h o m o d i m e r , w i t h t h ea c t iv e s i t e s s h a re d b e tw e e n th e s u b u n i t s , j u s t l i k e A A T , i t sp o ly p e p t id e c h a in i s o n ly 2 8 2 r e s id u e s lo n g a n d i t s tw od o m a i n s h a v e a to p o l o g y t h a t is c o m p l e t e l y u n r e l a t e d t oth o s e o f A A T (F ig u r e 2 ) [15 ,1 6 ].I n r e c e n t y e a r s , a t t e m p t s h a v e b e e n m a d e t o c la s s if y t h eB 6 e n z y m e s i n t o e v o l u t i o n a r y f a m i l i e s . D u e t o n o t o r i o u s l yw e a k s e q u e n c e i d en t it y , e v e n b e t w e e n B 6 e n z y m e s w i t hv e ry s im i l a r s p a t i a l s t ru c tu re s , t h i s w a s n o t a n e a s y t a s k ,a n d a c o m b i n a t i o n o f m o d e r n s e q u e n c e a l i g n m e n t a n ds e c o n d a r y - s t r u c t u r e p r e d i c t i o n m e t h o d s h a d t o b e a p p l i e d[1 6 -2 1 ] . C h r i s t e n a n d c o l l e a g u e s [1 8 ] d e f i n e d a n or , aa n d a y f a m i l y , n a m e d a f t e r t h e a t o m i n t h e a m i n o a c i d

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Key i n te rmed i a tes o f reac t i ons c a ta t y s ed byB6-de pende n t enz y mes . S t ruc tu re 1 , t hep ro tona ted i n te rna l a l d i m i ne be tween PLP andthe ac t i ve s i te l ys ine, i ll us t ra tes the cofa c tornome nc l a tu re us ed i n t h i s revi ew. T he m os tac t i v e f o rm o f t he p y r i dox a l d i m i ne has a cisoida l d i m i ne doub l e bond , a p ro tona ted py r i d i neN1 an d a dep ro tona ted 3 -hy d rox y l g roup .T rans a l d i m i na t ion w i t h t he am i no a c i ds ubs t ra te (w i t h an unc ha rged am i no g roup )p rodu c es t he ex te rna l a ld i m i ne . Ac c o rd i ng t oDuna than [ 52 ] , c on fo rma t i on 2a m ay lead t ot rans ami na t i on v ia t he qu i nono i d i n te rmed i a te3a , t he k e t i m i ne i n te rmed i a te 4a and i t shy d ro l y s is to t he ox o ac i d p rod uc t and P MP5a. This re ac t ion i s revers ib le . A lternati ve ly , 3ac a n b e r e p r o t o n a t e d f r o m t h e o p p o s i t ed i rec t i on t o g i v e t he ex te rna l a l d i m i ne 4d .T rans a l d i m i na t ion o f 4d p rodu c es t he D -aminoac i d p rodu c t and 1 aga i n ( rac emi s a t i on ) . [3 o rT e l i m i na t ion o f a X g roup f rom the Rs i dec ha i n a l s o s ta r t s w i t h 3a . I n t he ex amp l ei l lus t ra ted he re, a s i dec ha i n X -C H 2 -e l i m i na tes X , p ro duc i ng t he am i noac ry l a tei n te rmed i a te 4e . R ep ro tona t i on andt rans a l d i m i na t i on re fo rm 1 a nd t he i m i nep rod uc t s hown , wh i c h hy d ro l y s es t o py ruv a teand am mo nia. A l ternat ive ly , i n 13 rep la cem entreac t i ons , a g roup Y Q i s added t o C I ] ,res u l t ing i n t he qu i non o i d 5 f , wh i c h i srep ro ton a ted a t Cc ( t o t he ex te rna l a l d i m i ne 6 f.T rans a l d i m i na ti on res u l ts i n t he J ] rep l ac em en tp rodu c t and 1 . The ex te rna l a l d i m i ne 2b l eadsto ~ dec a rbox y l a t ion , p rodu c i ng t he qu i nono i d3b. Th is i s usual ly re pro tona ted at C(x form ingthe ex te rna l a l d i m i ne 4b and res u l t ing i n anamine produc t and 1. A l ternat i ve ly ,rep ro tona t i on a t C4 ' g i v es a k e t im i ne andf ina ll y an ox o p rodu c t and P MP(c o r res pon d i ng t o 3a -~ 4a -~ 5a ) . F i na l ly , t here l eas e o f t he who l e s i dec ha i n f rom 2 cp roduc e s , v ia qu i nono i d 3c and t he ex te rna la l d i m i ne 4c , t he g l y c i ne p rodu c t and 1 .

s u b s t r a t e a t w h i c h ( w i th f e w e x c e p t i o n s ) t h e c o v a l e n c yc h a n g e s t a k e p l a c e . P r o t o t y p e s o f t h e 0~ a n d ~ f a m i l i e s a r eA A T a n d T R P S [ 3 , r e s p e c ti v e l y . T h e s t r u c t u re o f C B L[ 14 ], d e t e r m i n e d a f t e r [ 1 8 ] a p p e a r e d , w a s t h e f i r s t r e p r e -s e n t a t i v e o f t h e 7 f a m i l y . T h i s s t r u c t u r e r e v e a l e d t h a t t h e? f a m i l y h a s t h e s a m e f o l d a n d , t h e r e f o r e , t h e s a m e a n c e s -t o r a s t h e o ~ f a m i l y. G o l d s m i t h a n d c o l l e a g u e s [ 21 ] h a dc o r r e c t ly c o m b i n e d t h e s e t w o f a m i l i e s i n t o t h e s o - c a l l ed' f o ld t y p e I ' . A c c o r d i n g t o t h i s n o m e n c l a t u r e , t h e T R P S 13f a m i l y is fo l d t y p e I I , w h e r e a s D A A T a n d r e l a t ed s t r u c -t u r e s r e p r e s e n t f o l d t y p e I V. F u r t h e r m o r e , t h e a u t h o r s [ 21 ]a ls o p r e d i c t e d t h a t e u k a r y o t i c O D C s a n d a l a n i n e r a ce -m a s e s h a v e a T I M - b a r r e l f o l d ( f ol d ty p e I I I ) . T h e r e c e n t l yp u b l i s h e d s t r u c t u re o f a l a n i n e r a c e m a s e ( A R ) f r omBacillus stearothermophilus [ 22 ] ( s e e b e l o w ) d e m o n s t r a t e st h e c o r r e c t n e s s o f t h is p r o p o s a l. G l y c o g e n p h o s p h o r y l a s e ,

w h i c h u s e s t h e p h o s p h a t e g r o u p o f P L P i n ca t a ly s i s ( a n dt h e r e f o r e i s n o t i n c l u d e d i n t hi s r e v i e w ) w a s l i s t e d u n d e rf o ld t y p e V . F o r s o m e f u r t h e r B 6 e n z y m e s o f u n k n o w ns t r u c t u r e , t w o a d d i t i o n a l f o l d t y p e s , V I a n d V I I , w e r e t e n -t a t i v e l y i n t r o d u c e d .I t s e e m s a p p r o p r i a t e f o r t h e p u r p o s e o f t h i s r e v i e w t o u s e af a m i l y c la s s if i c at i o n s y s t e m b a s e d o n t h r e e - d i m e n s i o n a ls t r u c t u r e s , u s i n g a s t h e f a m i l y n a m e t h e f i rs t r e p r e s e n t a -t i v e e n z y m e f o r w h i c h t h e s t r u c t u r e w a s d e t e r m i n e d( F i g u r e 2 ) . I t h u s d e f i n e t h e A A T , T R P S [ 3 , D A A T a n d A Rf a m i l i e s a n d w i l l d is c u s s n e w r e s u l t s o n e n z y m e s f r o mt h e s e f a m i l i e s i n t h a t o r d e r . T a b l e 1 l i s ts a l l t h e v i t a m i n B ~ ,-d e p e n d e n t e n z y m e s w h o s e t h r e e - d i m e n s i o n a l s tr u c t u r e sh a d b e e n p u b l i s h e d a t th e t i m e o f w r i t in g t h is r e v i e w a n di n t r o d u c e s t h e a b b r e v i a t i o n s u s e d h e r e .

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A s p a r t a t e D - am ino ac i da m i n o t r a n s f e r a s e a m i n o t r a n s f e r a s e A l a n i n e r a c e m a s eCur ren t Op in ion in S t ruc tu ra l B io log y

C a r t o o n p i c t u r e s o f e n z y m e s t h a t a r e r e p r e s e n t a t i v e o f t h e f o u rc u r r e n tl y k n o w n f o ld t y p e s f o r B B - d e p e n d e n t e n z y m e s . T h e v i e wd i r e c t i o n i s p a r a l l e l t o t h e m o l e c u l a r t w o f o l d a x e s . T h e P L P - l y s i n ea l d i m i n e s a r e d e p i c t e d i n b a l l a n d s t i c k m o d e . ( a ) T r y p t o p h a ns y n t h a e e o ~2 !3 2 c o m p l e x ( P D B c o d e 1 T T Q ) . C o l o u r c o d e : g r e e n ,c ( s u b u n i t s ; y e l l o w , [ 3 e u b u n i t 1 ; p u r p l e , N - t e r m i n a l d o m a i no f [3 s u b u n i t 2 ; a n d o r a n g e , C - t e r m i n a l d o m a i n o f [3 s u b u n i t 2 .( b ) A s p a r t a t e a m i n o t r a n s fe r a s e ( P D B c o d e 7 A A T ). C o l o u r c o d e :

y e l l o w , s u b u n i t 1 ; b l u e , N - t e r m i n a l s t r e t c h o f s u b u n i t 2 ; o r a n g e ,s m a l l d o m a i n o f s u b u n i t 2 ; a n d p u r p l e , l a r g e d o m a i n o f s u b u n i t 2 .( c ) D - a m i n o a c i d a m i n o t r a n s f e r a s e ( P D B c o d e 1 D D A ) . T h e c o f a c t o ri s P M P . C o l o u r c o d e : y e l l o w , s u b u n i t 1 ; o r a n g e , N - t e r m i n a l d o m a i no f s u b u n i t 2 ; a n d p u r p l e , C - t e r m i n a l d o m a i n o f s u b u n i t 2 . ( d ) A l a n i n er a c e m a s e ( P D B c o d e 1 S F T ). C o l o u r c o d e : y e l l o w , s u b u n i t 1 ; pu r p le ,N - t e r m i n a l d o m a i n o f s u b u n i t 2 ; a n d o r a n g e , C - t e r m i n a l d o m a i no f s u b u n i t 2 .

T h e a s p a r t a t e a m i n o t r a n s fe r a s e f a m i l yA m i n o t r a n sf e r a s es c a t al y s e a m i n o g r o u p t r a ns f e r b e t w e e na n a m i n o a c i d a n d a n o x o a c i d s u b s t r a t e i n t w o h a l f - r e a c -t io n s . T h e f i rs t f o l lo w s t h e p a t h w a y 1 - + 2 a - - + 3 a - + 4 a -+ S a

( F i g u r e 1 ). T h e s e c o n d p a s s e s t h r o u g h t h e s a m e i n t e r m e -d i a t e s in th e o p p o s i t e d i r e c ti o n . O n e s u b s t r a t e / p r o d u c t p a i ri s s p e c i f i c , t h e s e c o n d p a i r o f t e n i s 2 - o x o g l u t a r a t e / L - g l u t a -m a t e . M e h t a e t a . / . [ 1 7] d i v i d e d t h e a m i n o t r a n s f e r a s e s i n t o

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7 6 2 P ro t e i n s

T a b l e 1 . . .V i t a m i n B e - d e p e n d e n t e n z y m e s o f k n o w n t h r e e - d i m e n s i o n a l s t r u c t u r e .Enzyme Abbrevia t ion and EC number Source and re ferenceA s p a r t a t e a m i n o t r a n s fe r a s e ( A A T ) f a m i l y

07 family [18], fold type I [21]Asparta te aminotransferase

m-Amino acid :pyruvate aminotransferaseDia lky lg lyc ine decarboxylaseGlutamate-1-semia ldehyde am inomutaseOrn i th ine aminotransferaseTyrosine phe nol- lyaseTryptophan indo le-lyaseCystathionine 13-1yaseOrn i th ine decarboxylaseT ryp t o p h an syn t h ase I~ ( T R P S I ~ ) f am i l yfamily [18], fold type II [21 ]Tryptophan synthase ~ subuni tThreonine deaminaseO-acetylserine sulfhydrylaseD - am i n o ac i d am i n o t ran s f e rase ( D A A T ) f am i l yFold type IV [21]D-amino acid aminotransferaseBranched-chain amino acid aminotransferaseA l a n i n e r a c e m a s e ( A R ) f a m il yFold type II I [21]Alan ine racemase

AAT, EC 2.6 .1 .1

m-APT, EC 2.6 .1 .18DGD, EC 4.1.1 .64GSA T, EC 5.4 .3 .8OAT, EC 2.6 .1 .13TPL, EC 4.1 .99.2TIL, EC 4.1.99.1CBL, EC 4.4 .1 .8ODC, EC 4.1.1 .17

TRPSIS, EC 4.2 .1 .20TN, EC 4.2 .1 .16OASS, EC 4.2.99.8

DAAT, EC 2.6 .1 .21BCA T, EC 2.6 .1 .42

AR, EC 5.1.1 .1

Chicken m itochondria [4 ,26,27]Chicken cytoso l [5 ,25]Pig cytosol [5]E. co i l [24]S. cerevisiae [23]P s e u d o m o n a s sp. F-126 [8 ]mseudomonas cepacia [9,10,39]S y n e c h o c o c c u s [ 3 0 " ]Ho m o sa p i en s [31 ~32"]C. f r e u n d i i [11,36"]E. herbicola [37 ]P. vulgar is [38"]E. co i l [14,40,44]Lactobaci l lus 30a [12,13]

S. typhimurium [7,42",43,45]E. co i l [49"]S. typhimurium [51]

Baci l lus species [15,41 ]E. co i l [54]

B. stearothermophi lus [22"]EC, enzym e comm ission.

f o u r s u b g r o u p s a c c o r d i n g t o t h e i r e v o l u t i o n a r y r e l a t e d n e s s.A A T a n d D G D a r e, i n t h is c l a ss i fi c at i o n, r e p r e s e n t a t i v e s o fs u b g r o u p s I a n d I I , r e s p e c t i v e l y , w h i l e D A A T b e l o n g s t os u b g r o u p I I I .A c o m p a r i s o n o f th e n e w l y s o l v e d s t r u c t u r e o fSaccharomyces ce rev i s iae c y t o s o l i c A A T i n i t s m a l e a t ei n h i b i t e d c l o s e d f o r m [ 2 3 ] w i t h c o r r e s p o n d i n g s t r u c t u r e so f t h e Escher ichia co~ i , c h i c k e n c y t o s o l i c a n d c h i c k e nm i t o c h o n d r i a l e n z y m e s [ 2 4 - 2 7 ] r e v e a l e d h i g h s t r u c t u r a lc o n s e r v a t i o n , i n s p i t e o f c o n s i d e r a b l e e v o l u t i o n a r y d i s -t a n c e f r o m a l l t h r e e [ 2 3, 28 ] . T h e s t r u c t u r e o f A A T f r o ma n a r c h a e o n , f o r e x a m p l e , Sulfolobus sol fatar icus [29], isp r o b a b l y t h e o n l y o c c a s i o n w h e r e s i g n i f i c an t s t r u c t u r a ld i f f e r e n c e s c a n b e e x p e c t e d [ 2 8 ] .T w o n e w s t r u c t u r e s f r o m t h e a m i n o t r a n s f e r a s e s u b g r o u p I I[ 1 7 ] h a v e a p p e a r e d - - g l u t a m a t e - l - s e m i a l d e h y d e a m i n o -m u t a s e ( G S A T ) f r o m Synechococcus [3 0 ] a n d h u m a nr e c o m b i n a n t o r n i t h i n e a m i n o t r a n s f e r a s e ( O A T )[31,32 ,33]. Bo th e nz ym es hav e a fo ld tha t i s ve r y s imila rto th a t o f D G D [9, 10 ].T h e O A T s t ru c tu re , (x6 h e x a m e r ic ( in th e c ry s t a l a t l ea s t ) ,i s v e ry u n u s u a l i n th a t t h e th re e c a t a ly t i c d im e rs a re r e l a t -e d b y a n a p p r o x i m a t e t h r e e f o l d s c r e w a xi s, w i t h a

t r a n sl a t io n a l c o m p o n e n t o f 1 8 A [ 3 2 ]. L a c k o f O A T a c t iv -i t y d u e t o a m u t a t e d O A T g e n e c a u s e s g y r a t e a t r o p h y o fth e c h o ro id a n d r e t in a in h o m o z y g o te s , l e a d in g to b l in d -n e s s . T e n t a t i v e e x p l a n a t i o n s f o r t h e e f f e c t s o f s u c hm u t a t i o n s h a v e b e e n g i v e n [ 3 2 ] . T h e O A T s t r u c t u r e , a n di t s c o m p l e x e s w i t h t h e h y d r o x y l a m i n e d e r i v a t i v e L -c a n a l in e a n d th e s u ic id e in h ib i to r g a b a c u l in e [3 4 ] th ro wl ig h t o n th e c u r io u s d o u b le s p e c i f i c i ty o f O A T , w h ic ht r a n s a m i n a t e s t h e ( x - a m i n o g r o u p o f L - g l u t a m a t e a n d ,e x c lu s iv e ly , t h e 5 -a m in o g ro u p o f L -o rn i th in e [32 , 33 "] .A r g l 8 0 a n d T y r 5 5 p r o b a b l y b i n d t h e ( x c a r b o x y l a t e a n d t h e(x -a m in o g ro u p o f L -o rn i th in e , r e s p e c t iv e ly . T h e i r d i s p o s i -t i o n p r e v e n t s t h e b i n d i n g o f D - o rn i t h in e . A c o m p l e x w i t h( 2 S , 5 S ) - 5 - f l u o r o m e t h y l o r n i t h i n e , t h e o n l y i r r e v e r si b l ein h ib i to r a b s o lu te ly s p e c i f i c fo r O A T [3 5 ] , c o n f i rm s th ea b o v e p r o p o s a l ( P S t o r i c i , G C a p i t a n i , R M t i l l e r ,T S c h i r m e r , J N J a n s o n i u s , u n p u b l i s h e d d a ta ) . N o c o n f o r -m a t i o n a l c h a n g e s r e l a t i v e t o t h e u n l i g a t e d e n z y m e w e r eo b s e r v e d i n a n y o f t h e s e i n h i b i t o r c o m p l e x e s .G S A T , a h o m o d i m e r , c a t a l y se s t h e e x c h a n g e o f t h e a m i n oa n d o x o f u n c ti o n s o f g l u t a m a t c - l - s e m i a l d e h y d e ( G S A ). I t sa c t iv e fo rm i s t h e P MP -e n z y m e , w h ic h in a f i r s t h a l f - r e a c -t io n t r a n s fo rm s th e G S A s u b s t r a t e i n to 4 , 5 -d ia m in o v a le ra t e .T h e r e s u lt i n g P L P f o r m o f t h e e n z y m e t h e n t r a n s a m i n a te st h e 4 - a m i n o g r o u p o f t h i s f ir s t p r o d u c t a n d p r o d u c e s

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S tructure , evo lu t ion and act ion o f v i tamin B6-dependent enzyme s Jansonius 763

5-aminolevulinate (the building block in the biosynthesis of,for example, h eme and chlorophyll). Upon its release, thePMP--enzyme is left behind, ready to attack the next sub-strate molecule. The fact that GSAT is only found in plantsand certain bacteria makes it a potential target for safe,selective herbicides. The substrate-binding pocket wasidentified in a gabaculine complex. It has no open entrance,but disorder of a surface loop, residues 153-181, providesaccess to the binding site in one subunit, in which the PLPcofactor makes an aldimine linkag e with Lys273. In the sec-ond subunit, the 153-181 loop is structured and blocks theentrance to the active site, in which the cofactor is in thePMP form [ 30"]. A systematic investigation of this structur-al asymmetry is in progress. It is not an artefact of the crystalpacking, but is clearly related to function (J Stetefeld,JN Jansonius, unpublished data).TPL from C i t r o b a c t e r f r e u n d i i , a member of the {x family[18], is an {x4 tetramer with 222 symmetry, whose apoen-zyme structure is known [11]. The enzyme catalyses the13 elimina tion of phenol from L-tyrosine to produce py ru-vate and ammonia via the intermediates 1--+2a--+3a-+4e(Figure 1). Recently, the structure of the PL P- en zy me incomplex with the noncovalent inhibitor 3-(4'-hydrox-yphenyl) propionic acid was determined [36"]. A proposalfor the catalytic mechanism of TP L was made, based on thestructure and kinetic data for a variety of substrates of thewildtype enzyme and three Arg381 mutants. Catalytic roleswere attributed to the active site Lys257 (as usual), Arg381and Tyr71, while Arg404 (equivalent to Arg386 in AAT [4])is believed to bind the {x carboxylate of the substrate in adouble hyd rog en- bonded ion pair. In a parallel study onTPL from E r w i n i a h e r b i c o l a [37"], the structures of theapoenzyme, holoenzyme and the apoenzyme complexedwith the cofactor substrate analogue phosphopyridoxyl-L-tyrosine were solved and compared. The crystals used hadthe whole tetramer in the asymmetric unit and the relativedispositions of the two domains differed betweenmonomers. This led the authors to propose a slightly dif-ferent catalytic mechanism, with other roles for Arg381 andTyrT1. Further work is needed to resolve this issue.The structure of tryptophan indole-lyase (tryptophanase,TIL) from Proteus vulgar. is (unligated PLP-enzyme) hasalso appeared [38]. This enzyme, again a homotetramerwith dihedral symmetry, eliminates indole from L-trypto-phan in a reaction equivalent to that of TPL. It is a closerelative of TPL, with, for example, 50% sequence identitywith the C . f r e u n d i i enzyme. In this case, the four subunitsare also crystallographically independent and domainreorientations are observed. A K + ion binds, in both TP Land TI L, to each subunit, close to the interface of the 'c at-alytic' intimate dimer. It has an important structural roleand is essential for activity. If K + is replaced by N a +, bothenzymes are inactive [38"]. This situation is closely analo-gous to DGD, in which these ions have the same effect[9,10,39]. Th e K ion-b inding site in D GD i s , however,structurally distinct from those of TPL and TIL .

The structures of CBL from E. co l i and of a complexresulting from its suicide inhibition with trifluoro-L-ala-nine were published some time ago [14]. CBL, another{x4 tetramer with 222 symmetry, is the first representativeof the y family [18]. The main distinction between thisstructure and those of the {x family seems to be the foldand disposition of its N-terminal domain. A large part ofthis domain lies at the entrance of the active site of the sec-ond subunit of the 'catalytic' dimer and contributesresidues with a presumed catalytic function. While thefolds and relative positions of the PLP-binding and C-ter-minal 'small' domains are very similar in CBL, AAT andDGD, the N-terminal domain of CBL has a totally differ-ent fold and position. CBL catalyses the production ofhomocysteine, pyruvate and ammonia from cystathioninein a 13-elimination reaction equiva lent to those catalysed byTPL and TIL.Th e inhibition of CBL by L-aminoethoxyvinylglycine hasbeen investigated r ecently [40] using a combination of X-ray crystallography and kinetic methods. The end productis not covalently bound to the catalytic residue Lys210.Rather, a stable ketimine seems to be made that is kept inthe active site through hydrogen bonds and ionic interac-tions to active site residues. Neither the complex withtrifluoro-L-alanine nor that with L-aminoethoxyvinyl-glycine causes a conformational change of the enzyme,other than the usual cofactor tilt that was first observed inAAT [6] and, subsequently, in all covalent active site com-plexes of B 6 enzymes, regardless of their family[33",37",41"-43"]. Clausen e t a / . [44] believe, however,that protein conformational changes play a role in the cat-alytic mechanism with the natural substrate cystathionine,probably involving helix 3 in the N-terminal domain.Further experimental evidence for the postulated mecha-nism of action is needed.T h e t r y p t o p h a n s y n t h a s e 1~ f a m i l yThe TRPS { X 2 [ ~ 2 complex is a dimer of twofold-related (13{x)pairs, in which the 13 subuni ts lie back to back (Figu re 2)[7]. Th e two (13o0 dimers act independent ly, but each isallosterically regulated through its neighbouring subunit inthe dimer, such that subs trate binding to the {x subun itenhances the 13 reaction and vice versa. T he ot subunit, aTIM-barrel enzyme, produces glyceraldehyde-3-phosphateand indole from indoleglycerolphosphate (IGP). The13 subunit is a PLP enzyme, catalysing the 13-replacementreaction L-serine + indole--+ t.-tryptophan + H 20. Th ereaction follows the pathway 1--+2a--+3a--+4e--+5f--+6f(Figure 1), in which X and Y are OH and indole, respec-tively. In the 13-active site, the external aldimine withL-serine eliminates water to produce the aminoacrylate(A-A) intermediate (4e in Figure 1). Indole is stereospecif-ically added to this, resulting in L-tryptophan. The mostunusual feature of the total reaction is that indole passesthrough a 25 A long hydrophobic tunnel from the {x-activesite to the 13-active site. Over t he years, kine tic studies onwildtype TRPS and many mutant forms have identified

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7 6 4 P r o te i ns

residues that are critical for catalysis or for the allostericeffects. Recent crystal structures have led to more insightinto what happens at the atomic level [42,43,45,46].TRPS]3 is yet another enzyme with an alkali-ion-bindingsite that influences catalysis. Diffe rences in ligand bindingto a K + ion or a Na + ion at this site are t ransmitted as con-formational changes that cause ]3Tyr279 and 13Phe280 toeither line (when K + is bound) or block the tunnel (whenNa is bound). Thus, Na is inhibito ry [45].]3Lys87 is the PLP binding and catalytic lysine. In a]3K87T mutant, the PLP-L-serine and PLP-L-trypto-phan external aldimines are stable. The crystalstructures of these external aldimine intermediates ofthe 0tz]3z 13K87T mut ant comp lex (in the Na+-inhib itedform) have been determined. Two further structuresthat, in addition to L-serine in the ]3-active site, hadbound either the inhibitor indole-3-propanol phosphate(IPP) or the inhibitor D,L-~-glycerol-3-phosphate (GP)in the m-active site, were also elucidated [42"]. Thecombined structures provide a wealth of information.Space limitation prevents a detailed discussion of theconformational changes observed. Briefly, binding of L-serine causes a kind of 'subdomain closure' in the]3 subunit, but has no effec t on the 0~ subun it. When ,however, IPP or GP are also bound, the c~-active siteentra nce is blocked and the c~ subun it rotates 5 withrespe ct to the ]3 subunit. Th e bindin g of IPP alone didnot have this effect [7]. On the other hand, binding of L-tryp topha n by itse lf can also cause th e 5 rotation. Th etoo large distance between the presumed catalyticresidue otGlu49 and the inhibitors IPP and GP in the cor-resp ondin g ~K87T muta nt c~213e comp lex structure waspuzzling [42]. Mutation of the other catalytic residue,~Asp60, to asparagine (c~D60N) inacti vates the 0~ sub-unit. The substrate IGP was diffused into an ~213zc~D60N mutant crystal and X-ray data were collected at95K. The resulta nt struc ture had the y carboxyla te of~Glu49 in the expected productive position [46].Another group used an alternative, complementaryapproach [43], m aking use of the relative stability of theA-A int erm ediate in tile 13 reacti on in the a bsence o findole. A nat ive o~2132 com plex crystal, with 5-fluoro-indole propanol phosphate (F-IPP) bound in the cz-activesite, was mounted in a flow cell. By passing a (stabilising)solution of 200 mM L-serine through the flow cell, asteady state of the A-A intermediate was realised and X-ray data for the TR PS -F -I PP -A -A complex crystal couldbe collected and its structure determined. The nativeTRPS and the TRPS-F-IPP structures were also deter-mined, for comparison [43]. The TRPS-F-IPPstructure revealed that F-I PP (chosen because of parallel19F experiments) binds just like IPP itself. Fewerresidues were disordered in the c~ subunit than in theoriginal st udy with IPP [7], while o~Glu49 and ~Asp60were found in 'productive' positions and conformations.

In the ]3 subunit, there was also increased order, com-pared to the unligated enzyme, in two regions within asubdomain of the N-terminal domain (residues]3109-13189), which the authors named the COMMdomain. In their opinion, this is the unit that, throughrigid-body movement, communicates ligand-bindingeffects from one subunit to the other. One importanteffect caused by F-IPP binding is bringing the putativecatalytic residue ]3Glu109 1.5 A closer to the cofactor,which might be the origin of the allosteric activationobserved in solution.In the TRP S- F- IP P- A- A structure, the A-A externalaldimine is tilted by 20 , compared to 10 for the l.-serineexternal aldimine of the ]3K87T mutant. When comparedto the TRPS-F-IPP structure, the COMM domain is dis-placed slightly towards the C-terminal domain. This hassome small effects on the lengths ofintersubunit hydrogenbonds, one of which involves ~Asp60. The authors specu-late that this may activate the ~ reaction.All in all there are quite a few discrepancies between theresults of the two groups and their interpretation. Thesemay, however, be caused by the fact that, in one case, thewildtype enzyme, and, in the other, the ]3K87T mutantwas used, as well as by the different structures of the I.-ser-ine and A-A external aldimines. Although significantprogress has been made, further work is needed before thecomplicated catalytic mechanism of TRPS is fully under-stood. Mutagenesis experiments that probe specificstructural and functional details of this fascinating systemcontinue [47,48].The recently determine d structure of the allostericallyfeedback-regulated biosynthetic threonine deaminase(TN, also often called threonine dehydratase) from E. coli[49 ] is the second representative of the TRPSI3 family. Itproduces, through 13 elimination of a hydroxyl group, 2-oxobutyrate from L-threonine and pyruvate from I.-serine(compare to TRPSI3). The enzyme is an ~4 tetramer, adimer of 'catalytic' dimers. Each subunit consists of an N-terminal PLP-dependent catalytic domain and aC-terminal regulatory domain, connect ed by a helical neck.The subunits of the 'catalytic' dimer interact closely but,through a twisting of the neck, intersubunit, rather thanintrasubunit, contacts are formed between the N-terminaland C-terminal domains. The catalytic domain has a foldthat closely corresponds to that of TRPS]3 (root meansquare deviation of 1.8 A for 277 aligned o~-carbon atoms).Th e sequence identity level is marginal, however. The tun-nel wall residues in TRPS[3 have a different conformationin TN, which causes the tunnel to disappear. The cofactorPLP is bound to the active site Lys62 through an aldiminelinkage. As in TRPS]3, the PLP pyridine N1 (Figure 1) ishydrogen bonded to a serine, Ser315. The negative chargeon the 3' oxygen of PLP is stabilised by a hydrogen bonddonated by Asn89. The equivalent residue in TRPSI3,Gln114, does not seem to make such an interaction.

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Structure, evolution and action of vitamin Bs-depend entenzym es Jansonius 765

T h e p r e s e n t T N s t r u c t u r e is u n l i g a t e d , b u t a c o m p a r i s o nw i t h t h e s t r u c tu r a l l y v e r y s i m i l a r r e g u l a to r y s e r i n e - b i n d i n gd o m a i n f r o m p h o s p h o g l y c e r a t e d e h y d r o g e n a s e [ 5 0] su g -g e s t s a l o c a t io n fo r t h e a l lo s t e r i c s i t e t h a t c o r re l a t e s w e l lw i t h t h e r e s u lt s o f m u t a t i o n e x p e r i m e n t s [ 49 "] .T h e t h i r d s t r u c t u r e o f a m e m b e r o f t h e T R P S I 3 f a m i ly , t h a to f 0 - a c e t y l s e r i n e s u l f h y d r y l a s e ( O A S S ) f r o m Salmonellalyphimurium, an (xz d im e r , a l s o h a s h ig h s t ru c tu ra l s im i l a r i -t y , b u t l o w s e q u e n c e s i m i l a r i t y w i t h T R P S I 3 . T h eN - t e r m i n a l r e g i o n o f T R P S I 3 , i n c l u d i n g h e l i c e s 1 a n d 2 , isa b s e n t i n O A S S . A l s o , t h e lo n g lo o p b e tw e e n 13 s t r a n d s 8a n d 9 i n T R P S ~ t h a t c o n t r i b u t e s m u c h t o t h e i n t e r f a c ew i th th e 0 t s u b u n i t [7] is m u c h s h o r t e r i n O A S S a n d h a s ad i f f e r e n t c o n f o r m a t i o n [ 51 ].T h e D - a m i n o a c i d a m i n o t r a n s f e r a s e f a m i l yD A A T ( f r o m a t h e r m o p h i l ic Bacillus s p e c ie s ) w a s th e f i r s tm e m b e r o f th e a m i n o t r a n s f e r a s e s u b g r o u p I I I [ 1 7] or P L P -d e p e n d e n t e n z y m e f o ld t y p e I V [ 21 ] f o r w h i c h a st r u c t u r ew a s p u b l i s h e d [ 15 ]. T h e e n z y m e i s a h o m o d i m e r w i t h 2 82r e s i d u e s p e r c h a i n , c o n s i d e r a b l y l e ss t h a n t h e a m i n o t r a n s -f e r a s e s o f t h e t h r e e o t h e r s u b g r o u p s [ 17 ]. T h e m o n o m e rh a s a u n iq u e fo ld , w i th b o th o ~ /~ a n d o ~+13 fe a tu re s( F i g u r e 2 ). T h e P L P c o f a c t o r i s b o u n d t h r o u g h t h e u s u a la l d i m i n e l i n k a g e t o L y s 1 4 5 f r o m t h e C - t e r m i n a l d o m a i n( r e s i d u e s 1 2 1 - 2 8 2 ) a t t h e i n t e r f a c e w i t h t h e N - t e r m i n a l' s m a l l ' d o m a i n ( r e s i d u e s 1 - 1 2 0 ) [ 4 1 " ] . R e s i d u e s f r o m b o t hd o m a i n s a n d t h e s m a l l d o m a i n o f t h e n e i g h b o u r i n g s u b u n i tc o n t r ib u te to th e a c t iv e s i t e . U p o n d im e r i s a t io n , a l o n gl o o p f r o m t h e s e c o n d m o n o m e r a p p r o a c h e s t h e f i r s t a c t i v es i t e a n d c o n t r ib u te s A rg 9 8 * to i t ( s e e b e lo w ; a s t e r i s k s a reu s e d in th i s r e v ie w to in d ic a t e r e s id u e s f ro m th e s e c o n ds u b u n i t o f t h e c a t a ly t i c d in a e r ).T h e s t r uc t u r es o f t h e P L P - b o u n d f o r m an d o f t h e a p o e n -z y m c o m p l e x e d w i t h p h o s p h o p y r i d o x y l - D - a l a n i n e(P P L -D -A Ia ; a c o fa c to r s u b s t r a t e a n a lo g u e p ro d u c e d b yr e d u c i n g th e a l d i m i n e d o u b l e b o n d b e t w e e n P L P a n d D - al a-n in e ) p ro v id e d th e s t ru c tu ra l b a s i s fo r a m e c h a n i s t i c p ro p o s a l[4 1 " ' ] . T h e s u b s t r a t e a n a lo g u e d o e s n o t i n d u c e a n y d o m a inre o r i e n tat io n . T h e c o fa c to r p h o s p h a te i s f i x e d th ro u g h s e v e nh y d ro g e n b o n d s d o n a te d b y th e p ro te in m a t r ix , in a w a y th a tis ve ry . s imila r to tha t se en in AA T [6] . Glu17 7 is hydr oge nb o n d e d t o t h e p r o t o n a t e d P L P p y r i d i n e N 1 . T h e p y r i d i n e0 3 ' h a s n o d i r e c t p ro te in l i g a n d . U p o n th e lo s s o f t h ea ld im ine b ond to Lys145 , the cofac to r ti l t s fo rward (aga in , a ss e e n in A A T, p iv o t in g a t N 1 a n d th e p h o s p h a te [6] ) b y 2 0 int h e u n l i g a t ed P M P - b o u n d f o r m a n d e v e n b y 3 5 i n t h e s u b -s t ra te ana logue complex . In the la t te r , a s we l l a s in thein t e rn a l a ld im in e , t h e C 4 ' - N b o n d i s o r i e n te d cisoid, th en i t ro g e n b e in g c lo s e to 0 3 ' ( s e e F ig u re 1 ) . T h i s o r i e n ta t io nh a s s o f a r c o n s i s t e n t ly b e e n fo u n d in P L P a ld im in e s . T h ea la n in e c a rb o x y la t e o f P P L -D -A la m a k e s a d o u b le h y d ro g e nb o n d e d io n p ai r w i th A rg 9 8 * f ro m th e s e c o n d m o n o m e r .T h e u n i q u e f e a t u r e o f t h e a c t i v e s it e i s t h a t t h e A f a c e o fth e c o fa c to r [4 ] i s s o lv e n t e x p o s e d , w h e re a s in th e A A T a n d

T R P S I 3 f a m i l y e n z y m e s , t h e B f a c e i s e x p o s e d . T h e p o s i -t i o n o f A rg 98 * c o r re s p o n d s to th a t o f A rg 3 8 6 in A A T [4, 6] ,b e i n g n e a r e s t t o t h e 0 3 ' o f t h e c o f a ct o r o n t h e s i d e o p p o s i t et h e p h o s p h a t e g r o u p . A s a c o n s e q u e n c e , i n t h e e x t e r n a la l d i m i n e t h a t is m i m i c k e d b y t h e a n a lo g u e , t h e C a - H o f D -a la n in e ( in th e p r o d u c t iv e o r i e n ta t io n fo r p ro to n r e l e a s en o r m a l t o t h e c o n j u g a t e d p y r i d i n e - a l d i m i n e d o u b l e - b o n ds y s t e m [5 2 ] ) w i l l b e s i t u a t e d o n th e B fa c e . T h e a p r o t o nw i l l t h e re fo re b e c lo s e to th e e -a m in o g ro u p o f L y s 1 4 5 , t h ere s id u e p ro p o s e d to b c th e c a t a ly s t r e s p o n s ib l e fo r 0~ d e p ro -t o n a t io n a n d s u b s e q u e n t C 4 " p r o t o n a ti o n . T h u s , t h ere l a t iv e d i s p o s i ti o n s o f t h e c o fa c to r a n d th e s u b s t r a t e r e c o g -n i t io n s i t e A rg 9 8 * e x p la in th e s p e c i f i c i ty o f D A A T fo rD -a m in o a c id s. A ra th e r w id e p o c k e t b e h in d C I3 o f t h e s u b -s t r a t c a n a lo g u e a c c o m m o d a te s l a rg e r s id e c h a in s , i n l i n ew i t h t h e b r o a d s u b s t r a t e s p e c if i c i ty o f t h is e n z y m e . T h ero le o f A rg 9 8 * in s u b s t r a t e b in d in g h a s b e e n c o n f i rm e d b ys i t e - d i r e c t e d m u t a g e n e s i s [ 5 3 ] . T h e c a t a l y t i c m e c h a n i s mp ro p o s e d o n th e b a s i s o f t h e c ry s t a l s t ru c tu re s i s c o m p le t e -l y a n a l o g o us t o t h a t o f A A T . T h e s e t w o e n z y m e s i n d e e dp r o v i d e a s t r i k in g e x a m p l e o f c o n v e r g e n t e v o l u t i o n [ 15 ].B r a n c h e d - c h a i n a m i n o a ci d a m i n o t r a n s f e r a s e ( B C A T ) f r o mE . co l i e x h i b i t s 2 8 % s e q u e n c e i d e n t i t y w i t h D A A T a n d ,a c c or d in g l y, i s a m e m b e r o f t h e s a m e a m i n o t r a n s f e r a s es u b f a m i l y [ 1 7] a n d f o l d t y p e [ 21 ]. T h e h o m o l o g y o f t h e s et w o e n z y m e s , o n e s p e c i f i c fo r D -a m in o a c id s , t h e o th e r fo rL - a m i n o ac i ds , a n d t h e i r c o m m o n u n i q u e s t e r e o s p e c i f i c i tyo f p r o t o n t r a n s f e r a t p o s i t io n C 4 ' o f t h e c o f a c t o r ( F i g u r e 1 )[ 5 4 ] a r e i n t r i g u i n g . T h e r e c e n t l y d e t e r m i n e d s t r u c t u r e o fB C A T s u g g e s t s h o w t h i s e n z y m e m a n a g e s t o a c t o n s u b -s t r a te s o f o p p o s i t e c h i r al i ty c o m p a r e d t o D A A T [ 5 5 " '] .B C A T i s a n o t6 h e x a m e r w i t h 3 2 s y m m e t r y . T h r e e i n t im a t e' c a t a ly t i c ' d im e rs fo rm th e e d g e s o f a t r i g o n a l p r i s m , w i th ac y l in d r i c a l h o le s u r ro u n d in g th e th re e fo ld a x i s . T h e a c t iv es i t e s f a c e th e o u t s id e . T h e m o n o m e r , 3 0 8 r e s id u e s lo n g , i sfo ld e d l i k e D A A T , a n d th e c o fa c to r , b o u n d to L y s 1 5 9 , a l s oh a s th e s a m e p o s i t i o n a n d c o n fo rm a t io n , w i th i t s A fa c eb e i n g s o l v e n t e x p o s e d . R e s i d u e s f r o m t h e N - t e r m i n a ld o m a i n ( r e s i d u e s 1 - 1 2 6 a n d 3 0 3 - 3 0 8 ) , t h e l a r g e d o m a i n( r e s i d u e s 1 3 7 - 3 0 2 ) a n d t h e s m a l l d o m a i n o f t h e s e c o n ds u b u n i t c o n t r i b u t e t o t h e a c t iv e s it e . G l u 1 9 3 m a k e s a n i o np a i r w i t h t h e p r o t o n a t e d P L P p y r i d i n e N 1 . U n l i k e D A A T ,t h e 0 3 ' r e c e i v e s a d i r e c t h y d r o g e n b o n d , f r o m T y r 1 6 4 . T h ep u t a t i v e s u b s t r a t e - b i n d i n g r e s i d u e s a r e a l l d i f f e r e n t a sw e l l . T h e l . - a m in o a c id s u b s t r a t e m u s t , i n t h e e x te rn a la l d i m i n e i n t e r m e d i a t e , b i n d w i t h i t s ~ c a r b o x y l a t e o n t h es i d e o f t h e P L P p h o s p h a t e g r o u p , i n o r d e r fo r it t o p r e s e n ti t s 0 t p ro to n to L y s 1 5 9 a t t h e b a c k . T h e a u th o r s p ro p o s e , a st h e c a r b o x y l a t e - b i n d i n g s it e , t h e m a i n c h a i n N H g r o u p s o fT h r 2 5 7 a n d A l a2 5 8 , t o g e t h e r w i t h t h e p h e n o l i c h y d r o x y l o fT y r9 5 . O n th e o p p o s i t e s id e , P h e 3 6 , T y r3 1 * , V a i l0 9 * a n dT y r 1 6 4 f o r m a h y d r o p h o b i c b i n d i n g s i te f o r t h e s u b s t r a t es id e c h a in (v a l in e , i s o l e u c in e o r l e u c in e ) . A rg 9 7 is p ro p o s e da s th e m o s t p ro b a b le b in d in g s i t e fo r t h e o ) c a rb o x y la t e s o ft h e s e c o n d s u b s t r a t e p a i r L - g l u t a m a t e / 2 - o x o g l u t a r a t e .P r e l i m i n a r y u n p u b l i s h e d d a t a on t h e b i n d i n g o f 4 - m e t h y l -v a l e r a t e s e e m t o c o n f i r m t h e s e p r o p o sa l s .

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7 6 6 Proteins

D - A l a n i n e r a c e m a s e f a m i l yIn [21], fold type III corresponds to a superfamily includ-ing eukaryotic ornithine decarboxylases, bacterialdiaminopimelate decarboxylases and biosynthetic argi-nine decarboxylases (listed as group IV decarboxylases in[19]), as well as ARs. Goldsmith and colleagues proposethis fold type to be a (13/(~)8 (or 'T IM' ) barrel and theyprovide a quite detailed active site model for mouseODC, some features of which were subsequently con-firmed by site-directed mutagenesis. Even though thisproposal seemed convincing, the final proof, the three-dimensional structure, was lacking. This proof has nowbeen given by the recently published structure of ARfrom B. stearothermophilus (Figure 2) [22]. This enzymeturns out to be a homodimer of 388 amino acid residuesand one PLP, which is bound through an aldimine linkageto Lys39, per chain. The monomer folds into twodomains. The N-terminal domain, residues 30-240, con-sists of a largely canonical TIM barrel, which lacks onlyan (~ helix following the eigh th 13 strand. Th e C-te rminaldomain, mainly 13 type, co mprises residues 1-29 and241-388. The PLP cofactor lies on the C-terminal side ofthe TIM barrel. In the very compact dimer, with non-crystallographic twofold symmetry, a loop from theC-terminal domain of the second monomer, prominentlyincluding Tyr265*, contributes to the active site of thefirst monom er (Figur e 2). PLP is bound with its A face [4]exposed, just as in DAAT and BCAT. Its phosphate groupis fixed by seven hydrogen bonds donated by protein lig-ands and two water molecules (as counted in the model,Brookhaven Protein Data Bank [PDB] code 1SFT).Intriguingly, the pyridine N1 receives a hydrogen bondfrom Arg219 and thus must be unprotonated. The pre-sumably ionised 3-hydroxyl is proposed to be hydrogenbonded to Arg136, although the distance and relative ori-entation are not too favourable. An acetate ion is bound inthe active site. One carboxylate oxygen is hydrogenbonded to Arg136 NH1, the other to the mainchain nitro-gen of Met312* (of the second subunit). Th e authorspropose that the car boxylate of the substr ate binds in thesame way. They suggest that Lys39, released upon theformation of the external aldim ine with L-alanine, andTyr265*, which is located on the solvent-exposed side ofthe cofactor and is involved in a short hydrogen bondwith ND1 of His166, might be the deprotonation/proto-nation catalysts in a reaction following the intermediates1---~2a---~3a---~4d in F igure 1. Ster ically, it seems unlikely,however, that in an external aldimine with L-alanine, inwhich the c~ carboxylate is on the side of the pyridine 3'-hydroxyl, Lys39 can approach the o~ proton. Tyr265*seems to be better positioned for (~ deprotonation. Lys39would then have to reprotonate the quinonoid intermedi-ate, but would have to pick up a proton from somewherefirst. This issue is not yet settled, it seems.

C o n c l u s i o n sT h i s r e v i e w c o v e r s r e c e n t r e s u l ts o n t h e s t r u ct u r e a n dm e c h a n i s m o f e n z y m e s f r o m t h e fo u r c u r re n t ly k n o w n

families of B 6 enzy mes with evolutionarily distinct folds(illustrated in Figure 2). Accordingly, many different top-ics are discussed that are difficult to summarise. Oneimmediately obvious fact is the fast acceleration in theproduction of new data in this field. Although it was stillpossible to list all B 6 enzy mes of known thre e-di men-sional structure (Table 1), the fact that nearly half of theentries are based on papers from 1997 and 1998 suggeststhat such an undertaking will soon become difficult.Indeed, the numbe r of new structures of B 6 enzymesknown to this author to have been elucidated, but notpublished in time to be included here already approach-es 10 (four from the Biozentrum, University of Baselalone). The wealth of new data that will become availablein the near future should help to answer many open ques-tions concerning the structure/function relationships ofB 6 enzym es. For ex ample, the interactions that areresponsible for the strong binding of the cofactor (and thecovalent catalytic intermediates!) to the protein matrixvary widely. Th e numbe r of hydrogen bonds from proteinligands to the pho spha te group of PL P ranges from four[10] to eight [26,42 ] or nine [13] and the usually doubl enegative charge of this group ranges from being nearlycompletely compensated [14,15,26,37",38"] to hardlycom pen sat ed at all [10,22,30,32]. Is this 'ove rki ll' onone side or poor performance on the other? Why bother tostabilise the charge on the pyridine N1 through ahydrogen bond to an aspartate or glutamate[6,10,13,14,30,32,36-38,41 ,55 ] if a seri ne does thejob as well [7,49,51] and even an unproto nat ed N1 canbe tolerated, at least in a racemase [22]? Must the 3-hydroxyl group be ionised and what are the minimumrequirements for reaching that goal [6,22,38,41,42t?It would seem that computational methods will be need-ed for a systematic investigation of such issues. Anothertopic that needs to be investigated further is the mini-mum requirements for the productive binding of thesubstrate. Again, there seems to be room for a wide vari-ety of conditions. The beauty of the closed structure ofAAT, in which the dicarboxylate substrate makes doublehydrogen-bonded ion pairs with two arginincs [6,27],keeping the substrate in an optimum orientation foro~ dep rotona tion [52] (Fig ure 1, 2a--->3a) and, simu ltane -ously, completely occluded from solvent, has not beenencountered again in subsequent investigations on otherenzymes. Rather, the evidence seems to suggest thatmany (or even perhaps most) B6 enzymes do not need aclosed structure [10,30,32,41 ] and even do not alwaysneed a rigidly fixed substrate [10]. Is AAT an 'overe-volved' enzyme and, if so, what was it needed for? Theanswers to such questions may lie at a higher organisa-tional level, where interactions between enzymes and the'tuning' of metabolic pathways come into play.

What are the highlights of the research described here?Th e advances in understanding the mechanism of TR PS[42',43 "] certainly have to be mentio ned, even if muchremains to be done here. Another highlight is the insight

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S t r u c t u r e , e v o l u t i o n a n d a c t i o n o f v i t a m i n B s - d e p e n d e n t e n z y m e s J an s on iu s 7 6 7

gained into the remarkable flexibility of the active sitestructure of the DAAT fold [15] (Figure 2), whereby sim-ple sidechain replacements can make the transition from atrans amina tion cataly st for D-amino acids [14,41 '] to onefor L-amino acids [55"']. The remarkable convergent evo-lution that gave rise to this family and the AAT family oftransaminases, with great similarities in cofactor bindingand catalytic mechanism [41"], and the discovery of anew family of B6 enzymes with a TIM-barrel domain [22"]are further highlights. A curious phenomenon is the localdeviation of twofold symmetry in the me dimetic GSAT[30"], which must be related to function, although it isnot yet understood how. Many more surprises can beexpected, not only as a result of new structures from theknown B6 families, but also because further independentfamilies must exist [21,56]. No doubt, the approach ofstructural g enomics will play a significant role in identify-ing genes coding for novel B6 enzymes. A new era inresearch on the structure and function of vitamin B6-dependent enzymes has just begun.

N o t e a d d e d in p r o o fAfter completion of this review, three further relevant arti-cles appeared [57-59].A c k n o w l e d g e m e n t sT h a n k s a r e d u e t o U G r t i t t e r f o r p r e p a r i n g t h e m a n u s c r i p t , t o M J ~i gg i a n dT K n t ~ ch e l f o r t h e i r h e l p w i t h t h e f i g u r e s a n d t o W R M o n t f o r t f o r c r i t i c a l lyr e a d i n g t h e m a n u s c r i p t. I a m v e r y g r a te f u l to m y w i f e H e n n y f o r h e r s u p p o r ta n d u n d e r s t a n d i n g d u r i n g t h e p a s t 2 5 y e a r s i n B a s e l . F i n a n c i a l s u p p o r t f r o mt h e C a n t o n s B a s e l - S t a d t an d B a s e l l a n d a n d g r a n t s f r o m t h e S w i s s N a t i o n a lS c i e n c e F o u n d a t i o n f o r t h e r e s e a r c h c a r r i e d o u t in m y l a b o r a t o r y o v e r t h ey e a r s i s g r a t e f u l l y a c k n o w l e d g e d .

References a n d r e c o m m e n d e d readingPapers of particular interest, publ ished w ithin the annual period of review,have been highl ighted as: of special interest" ' of outstanding interest

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S t r u c t u re , e v o l u t i o n a n d a c t i o n o f v it a m i n B s - d e p e n d e n t e n z y m e s Ja n so n iu s 7 6 9

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amino t rans fe rase fo r C-4 ' hyd rogen t r ans fe r o f the coenzym e .J A m C h e m S o c 1993, 115:3897-3900.55. Okada K, Hirotsu K, Sato M, Hayashi H, Kagamiyama H: Three-* * d imens iona l s t r uc tu re o f E s c h e r i c h ia c o i l b ranched -cha in am inoac id amino t rans fe rase a t 2 .5 A reso lu t i on . J B iochem 1997,121:637-641.The structure of branched-chain amino acid am inotransferase is very similarto that of D-am ino acid am inotransferase (se e [41"' ]). The active sites arecompared and the binding sites for both the branched sidechain and the

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