Crystal Structure of Yeast Cytosine Deaminase Cytosine deaminase (CD) catalyzes the deamination of cytosine to uracil and that of 5-methylcytosine to thymine (Fig. 1). The antimetabolite 5-fluorouracil (5- FU) is one of the most active chemotherapeutic agents for cancer treatment, but it has limited efficacy due to gastrointestinal and hematological toxicities. Due to its ability to convert nontoxic 5-fluorocytosine (5-FC) into 5-FU and its absence in mammalian cells, the combination of 5-FC with CD in enzyme-prodrug gene therapy has been shown to effectively control tumor growth and is currently being evaluated in clinical trials. Here we have determined the yeast CD structure at 1.6 Å resolution at beamlines BL12B2 and BL41XU [1,2]. Fig. 1. Proposed catalytic mechanism for yeast CD. C 1JT α B α A α C α D α E α F β 1 β 2 β 3 β 4 β 5 N 1JT C 1UAQ N 1UAQ (a ( ) b) superfamily, whereas the 158-residue dimeric yeast counterpart is grouped into the CDA superfamily [3]. The active site of yeast CD contains one tightly bound zinc ion, which is tetrahedrally coordinated by His 62 , Cys 91 , Cys 94 , and a bound inhibitor (Fig. 3). The complex structure reveals that yeast CD converts the inhibitor 2-hydroxypyrimidine into 4-(R)hydroxyl-3,4- dihydropyrimidine, which is enantiomeric to the configuration observed in E. coli CD. Therefore, the crystal structures of bacterial and fungal CDs provide an excellent example of convergent evolution, in that they have evolved from unrelated ancestral proteins but have achieved the same deamination reaction. O O Zn 2+ N H O O H H O Glu64 O O - O O N H O O O O Zn 2+ O O H H N Cytosine Glu64 O N Glu64 Glu64 H H Uracil H N H Glu64 Zn 2+ NH 2 O - O - N H N H Zn 2+ NH 2 Zn 2+ NH 3 O - N H NH 2 Fig. 2. (a) The monomeric structure of yeast CD with the zinc ion shown as a magenta sphere with its ligands and the inhibitor (DHU) as ball-and-stick representations. (b) Structural superposition of yeast CD (red), B. subtilis CDA (blue), and the subdomain 2 of AICAR transformylase (green) [4]. The protein structure is composed of a central five-stranded β -sheet ( β 1- β 5) sandwiched by six α -helices ( α A - α F) (Fig. 2(a)). S urprisingly, even though yeast CD shares a higher se q uence identity to cytidine deaminases (CDAs), its closest structural match is the subdomain 2 of the A I CAR transformylase domain, due to the common α D helix and the same direction of the β 5 strand (Fig. 2(b)). The strong conservation of tertiary structures suggests that these enzymes are descendants of a single ancestral gene, and thereby define a new superfamily. I nterestingly, the 426-residue hexameric E. coli CD belongs to B ased on structural studies, we propose a deamination mechanism for yeast CD (Fig. 1). The substrate binds to an active site and induces a closed conformation to se q uester the reaction from the solvent. The water molecule activated by the zinc ion attac k s the C 4 atom with the assistance of G lu 64 serving as a proton shuttle. The product uracil moves toward the zinc ion for ligation and this would wea k en its interaction with the C-terminal tail, allowing its r e l e a s e f r o m t h e a c t i v e s i t e . T h e a c t i v e s i t e architectures of yeast CD and CDAs are stri k ingly similar. However, they contain uni q ue substrate- recognition residues, in particular, C-terminal tails. 22 (a) ( b) (a) ( b) References [1] Y.H. Hsu et al. : Acta Cry s t . D 59 (2003) 950 . [ 2 ] S .H. Liaw et al. : J . Biol . Chem . 278 (2003) 4957 . [ 3 ] G . C . Ireton et al. : J . Mol . Biol . 315 (2002) 687 . [ 4 ] T . -P . Ko, J . -J . Lin, C . - Y. Hu , Y. - H. Hsu , A .H. -J . Wang and S . - H. Liaw: J . Biol . Chem . 278 (2003) 1 9 111. Fig. 4. Molecular surfaces of yeast CD (A) and B. subtilis CDA (B) colored for electrostatic potentials from –10 k B T (red) to 10 k B T (blue). The C-terminal tail (residues 149-158) of yeast CD is shown explicitly as worms as for the C-terminal residues (residues 122 * -131 * ) from one adjacent subunit in B. subtilis CDA. The analogues for cytosine and cytidine are colored green and yellow, respectively. The CDAs have space to accommodate the ribosyl sugar of cytidine, whereas yeast CD does not have space due to the blocking by the C-terminal tail [4]. The pre s ence of an α D helix in yea s t CD lead s s trand s β 4 and β 5 to be parallel and thi s re su lt s in the C-terminal tail moving back s harply t o w a r d s t h e a c t i v e s i t e t o accommodate only the cyto s ine ba s e within the s ame molec u le (Fig . 4(a)) . On the other hand, the lack of an α D helix in B. subtilis CDA lead s s trand s β 4 and β 5 to be antiparallel and thi s re su lt s in an o p p o s i t e d i r e c t i o n f o r t h e C - terminal tail, which enlarge s the active s ite of the adjacent molec u le to accommodate a larger cytidine su b s trate (Fig . 4(b)) . P r e v i o u s s t u d i e s h a v e demon s trated that yea s t CD ha s a greater therape u tic potential than t h e b a c t e r i a l e n z y m e s i n t h e enzyme-prodr u g s trategy . The C 5 atom of the pyrimidine ring i s adjacent to a hydrophobic cl us ter and face s toward Phe 11 4 , with a di s tance of 4 .1 Å . When a fl u orine atom i s attached to an aromatic ring, it will make it very hydrophobic . Therefore, the hydrophobic cl us ter in yea s t CD s ho u ld enhance the binding of 5 -FC . On the other hand, the C 5 atom face s toward A s p 3 1 4 i n E . c o l i C D , w i t h a di s tance of 3 . 6 Å between C 5 and A s p3 1 4 O δ 1 . Clo s e contact with su ch a polar re s id u e wo u ld be u n f a v o r a b l e t o 5 - F C b i n d i n g . The s e s tr u ct u ral ob s ervation s are con s i s tent with enzyme kinetic mea su rement s. Shw u - Hu ey Liaw Fac u lty of Life Science, National Y ang-Ming Univer s ity, Taiwan E-mail: s hliaw@ym . ed u. tw Fig. 3. Active site. (a) The 2F o – F c map at 1.5 σ level in cyan, the difference anomalous map for the zinc ion at 30 σ m level in purple, and the densities for the inhibitor (DHU) in green. (b) The interaction networks in the active site. There are six direct hydrogen bonds between the protein and the inhibitor [4]. 23
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