Part:BBa_K404112

Cytosine deaminase (CD)

Usage and Biology

Cytosinde deaminase
BioBrick Nr.	BBa_K404112
RFC standard	RFC 25
Requirement	pSB1C3
Source	codA gene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Cytosine Deaminase (CD) (EC 3.5.4.1) in E.coli is encoded by codA and plays a crucial role in nucleotide synthesis due to deamination of cytosine to uracil. Subsequently, cytosine can be used for pyrimidine synthesis [Kilstrup et al., 1989, Anderson et al., 1989]. The expression of codA is regulated by the concentration of nitrogen in the media. In presence of either purines or pyrimidines, the expression is repressed, while lack of any nitrogen leads to overexpression of CD [Andersen et al., 1989].
The CD’s function is based on conversion of 5-Fluorocytosine (5-FCyt) to the highly toxic product 5-fluorouracil (5-FUra), which leads to cell death [Polak and Scholer, 1975]. CodA mutant E.coli as well as mammalian cells seem to be resistant to 5-FCyt due to the deficient metabolic activation [Beck et al., 1972, Hoeprich et al., 1974]. The resistance in E.coli arises because of the loss-of-function of cytosine permease codB, which is located adjacent to codA [Lind et al., 1973].

Figure 1: Fluorocytosine pathway. Adapted from Fluorcytosine pathway (Yoshiaki Adachi et al., 2000).

Due to this strong effect and the fact that mammalian cells do not express CD naturally [Koechlin et al., 1966], 5-FUra became a promising compound in suicide gene therapy. 5-FUra itself is metabolized to 5-fluorouridine-5‘-triphosphate (FdUTP) and 5‘-fluoro-2‘-deoxyridine 5‘-monophosphate (FdUMP) [Aghi et al., 1998], which in turn impair RNA as well as DNA synthesis and causes apoptosis [Mullen et al., 1992; Damon et al., 1989]. FdUMP inhibits thymidylate synthetase, which is responsible for catabolism of deoxyuridylate into thymidylate. Consequently, treatment of cells with 5-FUra leads to failure of thymidylate synthetase and subsequently loss of dTTPs. Hence, during the DNA synthesis, both FdUTP as well as uridine throphosphate become incorporated into the DNA leading to the nick in the stand. dTTPs plays also crucial roles in repair events, thus dTTPs depletion is associated with repair failure and cell death [Aghi et al., 1998]. Another advantage of 5-FUra is its solubility and ability to freely diffuse into adjacent cells [Domin et al., 1993; Huber et al., 1994] therefore enhancing the bystander effect. Also, 5-FUra might interfere in the function or maturation events of RNA molecules, such as mRNA polyadenylation, tRNA methylation and processing of rRNA [Aghi et al., 1998].

Figure 2: Crystal structure of the E. coli cytosine deaminase (PDB entry: 1k6w).