Crystal Structure of Yeast Cytosine Deaminase INSIGHTS INTO ENZYME MECHANISM AND EVOLUTION*

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-hydroxy-pyrimidine at 1.6-Å resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 Å 2 per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essen-tial zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His 62 , Cys 91 , and Cys 94 from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxam-ide-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