SUMMARY PARAGRAPH for FCY1
Fcy1p is a cytosine deaminase (EC:188.8.131.52) that catalyzes the hydrolytic deamination of cytosine to uracil (1, 2, 4). Fcy1p is homodimeric, and each subunit contains an active center composed of a catalytic zinc ion coordinated with a histidine (His62), two cysteines (Cys91 and Cys94), and a water molecule in the substrate-free enzyme, with the latter serving as a nucleophile in the deamination reaction (2). The conversion of cytosine to uracil is required for the pyrimidine salvage pathway of Saccharomyces cerevisiae, and is important for providing pyrimidines for the synthesis of nucleic acids and amino acids, and as a source of energy (4, 5). A block in Fcy1p cytosine deaminase activity, but not in Cdd1p cytidine deaminase activity, constitutes a limiting step in cytidine utilization (4). Fcy1p is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU) (2).
FCY1 is similar to Candida albicans (FCA1) and Escherichia coli cytidine deaminases (1, 2), and displays significant structural similarity to Bacillus subtilis cytidine deaminase (cdd) and chicken ATIC (6). Fcy1p also displays two regions of similarity to the dCMP/CMP deaminases from human (DCTD), S. cerevisiae (DCD1) and T4 phage (dCD), and to the human cytidine/deoxycytidine deaminase CDA (1).
Fcy1 null mutants are viable, but display a total loss of cytosine deaminase activity, high resistance to 5-fluorocytosine, and resistance to 5-fluorocytidine (4, 1, 5). Although wild-type Fcy1p shows a rapid loss of activity at 50 C, with a half-life of 4 hours in vitro, Fcy1p-[A23L/I140L/V108I] has a half-life of 117 hours at 50 C, a 30-fold increase (7). Fcy1p-[A23L/I140L/V108I] is also better than wild-type Fcy1p at allowing growth at 37 C of an E. coli strain of dependent on cytosine deaminase function for uracil synthesis (7). The defects of S. cerevisiae fcy1 mutants can be complemented by C. albicans FCA1 (1).
A novel and highly potent suicide gene, FCU1, designed for expression in tumorogenic mammalian cells, has been derived from S. cerevisiae FCY1 cytosine deaminase and FUR1 uracil phosphoribosyltransferase. The bifunctional chimeric Fcu1p combines the enzymatic activities of Fcy1p and Fur1p, and efficiently catalyzes the direct conversion of nontoxic antifungal 5FC into the toxic metabolites 5FU and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. The uracil phosphoribosyltransferase activity of Fcu1p is equivalent to that of Fur1p, but its cytosine deaminase activity is 100-fold higher than that of Fcy1p. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) are sensitive to concentrations of 5FC 1000-fold lower than those used for cells transduced with a vector expressing only FCY1 (Ad-FCY1). Furthermore, bystander cell killing is also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. In a mouse model of tumor progression, injection of Ad-FCU1 into tumors in conjunction with systemic administration of 5FC, leads to substantial delays in tumor growth (8).
Last updated: 2005-11-04