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 Å 2 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 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-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.Cytosine deaminase (CD, 1 EC 3.5.4.1) catalyzes the deamination of cytosine to uracil and 5-methylcytosine to thymine. The enzyme has been found in bacteria and fungi, where it plays an important role in pyrimidine salvage. However, it is not present in mammalian cells, which utilize cytidine deaminase (CDA) instead (1). The bacterial and fungal CDs are distinct from each other and have evolved separately. The 426-residue hexameric Escherichia coli enzyme like the murine adenosine deaminase belongs to the (/␣) 8 -barrel amidohydrolase superfamily, in which four histidines and one aspartate located at similar spatial positions are conserved for metal coordination and enzyme catalysis (2-4). On the other hand, the 158-residue dimeric yeast counterpart may share two conserved signature sequences, HXE and CXXC, with a variety of deaminases, and thus has been grouped into the cytidine and deoxycytidylate deaminase family in the Pfam protein family data base (5, 6). The crystal structure of E. coli CDA reveals that the signature sequences contain a zinc binding motif, with histidine and two cysteines acting as zinc ligands while the glutamate serves as a proton shuttle (7).The antimetabolite 5-fluorouracil (5-FU) is one of the most active chemotherapeutic agents for the treatment of colorectal cancer, but it has limited efficacy due to gastrointestinal and hematological toxicities (8). Because of its ability to convert the relatively nontoxic 5-fluorocytosine (5-FC) into 5-FU and its absence in mammalian cells, CD has become an attractive candidate for the reduction of 5-FU toxicity toward n...