title: Crystal structure of yeast cytosine deaminase. Insights into enzyme mechanism and evolution.
abstract: 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-A resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 A2 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.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.forename: Jing-Jer
authors.initials: JJ
authors.lastname: Lin
authors.forename: Chih-Yung
authors.initials: CY
authors.lastname: Hu
authors.forename: Yi-Hsin
authors.initials: YH
authors.lastname: Hsu
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
authors.forename: Shwu-Huey
authors.initials: SH
authors.lastname: Liaw
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D043525:Cytosine Deaminase; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D019143:Evolution, Molecular; D005437:Flucytosine / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism / Q000633:toxicity; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009700:Nucleoside Deaminases / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D011743:Pyrimidines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D011743:Pyrimidines; C041345:2-hydroxypyrimidine; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil