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-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.
Ko
Tzu-Ping
TP
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Lin
Jing-Jer
JJ
Hu
Chih-Yung
CY
Hsu
Yi-Hsin
YH
Wang
Andrew H-J
AH
Liaw
Shwu-Huey
SH
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
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D011743:Pyrimidines; C041345:2-hydroxypyrimidine; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil