Fig. 1. Overall structure of MutM. The MutM molecule consists of an N-terminal domain (blue), a C-terminal domain (red, orange and green) and two long loops (yellow). The N-terminal domain consists of a two- layered β β -sandwich with two alpha helices. The C-terminal domain consists of four α -helix bundles (red and orange) and a β -hairpin loop of the zinc finger motif (green). In aerobic organisms, cellular DNA is easily damaged by activated oxygen species resulting from aerobic energy metabolism or oxidative stress. H i g h l y r e a c t i v e o x y g e n a c c e l e r a t e s t h e spontaneous mutation rate and therefore has been implicated as a causative agent for aging or the pathogenesis of disease, including cancer. One of the most common products of oxidative DNA damage is the 8-oxoguanine (GO) lesion. GO can pair with cytosine (C) as well as adenine (A), causing conversion from guanine (G) to thymine (T). To prevent mutation, the MutM protein removes GO bases from GO:C pairs in DNA. The mutM ( fpg ) gene encoding the MutM protein is highly conserved across a wide range of aerobic CRYSTAL STRUCTURE OF A REPAIR ENZYME OF OXIDATIVELY-DAMAGED-DNA, MutM (Fpg), FROM AN EXTREME THERMOPHILE, Thermus thermophilus HB8 1 7 8 6 4 3 5 9 2 B A F C E D Zn 2+ C-ter. Pro1 N-terminal domain C-terminal domain 1 0 1 1 N-ter. 1 7 8 6 4 3 5 9 2 B A F E D 10 C N-terminal domain C-terminal domain 11 Zn 2+ bacteria. These enzymes ( Mr= 30 kDa) possess the invariant N-terminal sequence Pro-Glu-Leu-Pro- Glu-Val-, two strictly conserved lysine residues (Lys52 and Lys147), and a zinc finger motif (-Cys- X2-Cys-X16-Cys-X2-Cys-) at the C-terminus. We determined the structure of the MutM enzyme derived from an extremely thermophilic bacterium, Thermus thermophilus HB8 at 1.9 Å resolution using MAD phasing of the intrinsic Zn 2+ ion of the zinc finger at beamline BL45XU [1-4]. The crystal structure of MutM comprises two distinct domains and a new fold connected by a flexible hinge (Fig. 1). Two molecules are detected in an asymmetric unit within the crystal. The overall conformations of the two independent molecules Zn 2+ minor groove dsDNA with C:GO pair major groove DNA backbone flipped-out nucleotide A 4 3 5 9 7 2 8 C G O H2TH catalytic site zinc finger motif turn β 5- β 6 6 F C D 11 10 E 6 Zn 2+ turn β 8- β 9 9 (I) ( II ) ( III ) ( IV ) A B Fig. 2. (A) The model of the MutM - DNA complex between the flipped-out DNA and MutM in the closed fo rm wa s ob ta in ed by mo le cu la r dy na mi c ca lc ul at io n. Th e ki nk ed DN A is dr aw n as a CP K mo de l wi th backbones colored in steel blue and with bases in steel gray. C and GO bases and their sugar residues before and after flipping out are shown by ball-and-stick models. All the four conserved regions ( I-IV ) are in the large cleft of the MutM molecule. The N-terminal domain has access to the major groove of DNA and the zinc finger motif of the C-terminal domain to the minor groove. The H2TH motif of the C-terminal domain is situated near the active site, and may interact with the damaged base of the DNA backbone. (B) There are two con for mer s in an asy mme tri c uni t. The con for mat ion s of the two lon g loo ps in the int er- dom ain cle ft differ between the two conformers. These two long loops would work as a hinge in domain movement. i n d i c a t e t h e e x i s t e n c e o f s i g n i f i c a n t h i n g e movem ent, where as the indiv idual domai ns of the t w o m o l e c u l e s i n t h e a s y m m e t r i c u n i t a r e s u b s t a n t i a l l y s i m i l a r ( F i g . 2 B ) . A l a r g e , e l e c t r o s t a t i c a l l y p o s i t i v e c l e f t l i n e d b y h i g h l y co ns er ve d re si du es e xi st s be tw ee n th e do ma in s. Pro1 Glu5 Glu 2 Lys52 α E α F α A β 3 β 4 H O 2 Pro1 Lys52 Glu2 Glu5 N + 5’ Lys52 Glu2 Glu5 3’ Pro1 C 1 ’ GO O8 A B C H O 2 H O 2 H O 2 5’ C 5 ’ C 4 ’ C 2 ’ C 3 ’ C 1 ’ Fig. 3. Archi tectu re of the activ e site of MutM. (A) The nucle ophil e Pro1 of MutM is surro unded by the invariant charged residues Glu2, Glu5 and Lys52, accompanied by several bound water molecules. (B) Model o f d o c k i n g o f t h e f l i p p e d - o u t G O n u c l e o t i d e t o M u t M a c t i v e s i t e b a s e s o n t h e d s D N A c o m p l e x . ( C ) T h e p u t a t i v e r e a c t i o n i n t e r m e d i a t e a d d u c t w i t h P r o 1 a f t e r β - e l i m i n a t i o n , w h i c h i s w e l l d e f i n e d d u e t o i t s conjugated double bond and can fit to the active site; the hydroxyl group at C4’ of the opened deoxyribose reaches to the carboxylic acid of Glu2, which is a good candidate for proton acceptor. Based on previous biochemical experiments and th e th re e- di me ns io na l st ru ct ur e, we co ns tr uc te d a s t r u c t u r a l m o d e l o f t h e d s D N A - M u t M c o m p l e x ( F i g s . 2 A a n d 3 ) a n d p r o p o s e a n e w r e a c t i o n mechanism for MutM ( Fig. 4 ). The locations of the putative catalytic residues and the two DNA-binding B Mitsuaki Sugahara a* and Seiki Kuramitsu a,b (a) Osaka University (b) SPring-8 / RIKEN * p resent address : SPring-8 / RIKEN E-mail: sugah@spring8.or.jp Fig. 4. Schematic representation of the r e a c t i o n m e c h a n i s m o f M u t M N - glycosylase/AP-lyase. We propose that the invariant amino acid residues (Glu2, Glu5, and Lys52) are in the vicinity of the primary catalytic residue Pro1. In this h i g h l y e l e c t r o s t a t i c a l l y p o s i t i v e environment, Lys52 may act as a proton donor for depuration of the damaged base (Fig. 3B). After the C2’ of deo xyribose forms a Schiff base with Pro1, Glu5 can withdraw the proton of C2 ’ via a bound water molecule, leading to β β -elimination. The resulting adduct intermediate (Fig. 3C) would deprotonate at C4 ’ of the opened deoxyribose, leading to δ -elimination. Finally, the recapture of the proton by Lys52 would release the other product, 4- oxo-2-pentenal, to form the gapped dsDNA product. The residues that will contribute to each reaction step were deduced from the crystal structure and are shown in red. motifs (the zinc finger and the helix-two turns-helix motifs) suggest that the oxidized base is flipped-out f r o m d o u b l e - s t r a n d e d D N A a n d e x c i s e d b y a catalytic mechanism in a manner similar to that of b i f u n c t i o n a l b a s e - e x c i s i o n - r e p a i r e n z y m e s . T h i s m o d e l d e t a i l i n g t h e f o r m a t i o n o f a d s D N A - M u t M c o m p l e x a c c o u n t s f o r t h e m u l t i p l e e n z y m a t i c activities assigned to MutM. The DNA glycosylase e x c i s e s v a r i o u s d a m a g e d b a s e s f r o m D N A b y forming a covalent Schiff base intermediate, which is for med at the dam age d sit e by the nuc leo phi lic attack of the Pro1 secondary amino group at the N- t e r m i n u s o f t h e d e o x y r i b o s e C 1 ’ t o p r o d u c e a n aldehydic abasic site. The AP lyase cleaves the 3’- p h o s p h o d i e s t e r b o n d a t A P s i t e s t h r o u g h β β - e l i m i n a t i o n . I n a d d i t i o n , a n a l t e r n a t i v e A P l y a s e c l e a v e s t h e 5 ’ - p h o s p h o d i e s t e r b o n d t h r o u g h δ δ - e l i m i n a t i o n . T h e s e r e a c t i o n m e c h a n i s m s a l s o e x p l a i n t h e d i f f e r e n t N - g l y c o s y l a s e / A P - l y a s e activities among MutM, E. coli endonuclease III and T 4 e n d o n u c l e a s e V . T o c o n f i r m t h e d e t a i l e d m e c h a n i s m o f t h e M u t M N - g l y c o s y l a s e / A P - l y a s e r e a c t i o n s , m u t a t i o n a l a n a l y s e s o f t h e a c t i v e - s i t e residues are currently undertaken. References [1] T. Mikawa et al ., Nucleic Acids Res. 26 (1998) 903. [2] M. Sugahara et al ., J. Biochem. 127 (2000) 9. [ 3 ] M . S u g a h a r a , T . M i k a w a , T . K u m a s a k a , M . Yamamoto, R. Kato, K. Fukuyama, Y. Inoue and S. Kuramitsu, EMBO J. 19 (2000) 3857. [ 4 ] M . Y a m a m o t o e t a l . , J . S y n c h r o t r o n R a d . 5 (1998) 222.
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