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J. Biol. Chem., Vol. 278, Issue 21, 19111-19117, May 23, 2003

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Crystal Structure of Yeast Cytosine Deaminase

INSIGHTS INTO ENZYME MECHANISM AND EVOLUTION^*

Tzu-Ping Ko  , Jing-Jer Lin  ¶, Chih-Yung Hu ||, Yi-Hsin Hsu ¶, Andrew H.-J. Wang  and Shwu-Huey Liaw || **  

From the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ^¶ Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei 11221, Taiwan, ^|| Institute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan, ^** Department of Life Science, National Yang-Ming University, Taipei 11221, Taiwan, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan

Yeast cytosine deaminase is an attractive candidate for anticancer gene therapy because it catalyzes the deamination of the prodrug 5-fluorocytosine to form 5-fluorouracil. We report here the crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine at 1.6-Å resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 Ų per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essential zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His⁶², Cys⁹¹, and Cys⁹⁴ from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase and thereby may define a new superfamily. The unique C-terminal tail is involved in substrate specificity and also functions as a gate controlling access to the active site. The complex structure reveals a closed conformation, suggesting that substrate binding seals the active-site entrance so that the catalytic groups are sequestered from solvent. A comparison of the crystal structures of the bacterial and fungal cytosine deaminases provides an elegant example of convergent evolution, where starting from unrelated ancestral proteins, the same metal-assisted deamination is achieved through opposite chiral intermediates within distinctly different active sites.

Received for publication, January 27, 2003 , and in revised form, March 11, 2003.

To whom correspondence should be addressed: Dept. of Life Science, National Yang-Ming University, Taipei, Taiwan 11221. Tel.: 886-2-2826-7278; Fax: 886-2-2820-2449; E-mail: shliaw{at}ym.edu.tw.

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