| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 91 === Cytosine deaminase (CD, 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 the cytidine deaminase instead. Due to the ability to convert the relatively nontoxic 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and its absence in mammalian cells, cytosine deaminase becomes an attractive candidate in the enzyme-prodrug gene therapy. Currently, combination of 5-FC with cytosine deaminases has proven effective at controlling tumor growth in animals and is being evaluated in several human clinical trials. In this thesis, the yeast cytosine deaminase is subject for structural studies because it has higher catalysis activity than that of bacterial enzymes. The recombinant enzyme is expressed in E. coli and purified using Ni-column. Hanging-drop vapor diffusion method was used to crystallize the purified protein. The crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine was diffracted with the resolution of 1.6 Å. The protein forms a tightly packed dimer with an extensive interface of 1450 Å2 per monomer. The inhibitor is 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 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.
Because of the catalytic subunit of human telomerase gene, hTERT, is expressed in most of the tumor cells but not in normal cells, we applied the hTERT promoter to direct yeast CD expression in cancer cells. This type of construction should provide selective and effective killing of cancer cells. However, because the expression level of yeast CD is low in tumor cells, the hTERT promoter driven yeast CD system can moderately enhance the toxicity of H1299 cells upon 5-FC treatments.
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