Cytosine deaminase

With the aid of cytosine permease, flucytosine reaches the fungal cell where it is converted by cytosine deaminase into 5-fluorouracil [51-21-8]. Cytosine deaminase is not present in the host, which explains the low toxicity of 5-FC. 5-Fluorouracil is then phosphorylated and incorporated into RNA and may also be converted into 5-fluorodeoxyuridine monophosphate, which is a potent and specific inhibitor of thymidylate synthetase. As a result, no more thymidine nucleotides are formed, which in turn leads to a disturbance of the DNA-synthesis. These effects produce an inhibition of the protein synthesis and cell repHcation (1,23,24). 5-Fluorouracil caimot be used as an antimycotic. It is poorly absorbed by the fungus to begin with and is also toxic for mammalian cells. 

Flucytosine-resistant strains can develop very rapidly. These mutants may have a disturbed 5-FC-metabohsm, or a compensatory mechanism for the disturbed nucleic acid functions. No cytosine permease was found in a resistant Cyptococcus neoformans strain, whereas cytosine deaminase was absent in resistant C. albicans strains. A deficiency of uridine monophosphate pyrophosphorylase occurs frequently in resistant C. albicans strains (1). 

Many yeasts are inhibited by 5-fluorocytosine and a block in the synthesis of 5-fluorouridylic acid by loss of cytosine deaminase or of nracil phosphoribosyltransferase is sufficient to cause resistance. Mntational loss of pyrimidine salvage enzymes has been frequently observed. 

A common way to benefit from the ability to combine different molecular orbital methods in ONIOM is to combine a DFT or ab-initio description of the reactive region with a semi-empirical treatment of the immediate protein environment, including up to 1000 atoms. Due to the requirement for reliable semi-empirical parameters, as discussed in Section 2.2.1, this approach has primarily been used for non-metal or Zn-enzymes. Examples include human stromelysin-1 [83], carboxypeptidase [84], ribonucleotide reductase (substrate reaction) [85], farnesyl transferase [86] and cytosine deaminase [87], Combining two ab-initio methods of different accuracy is not common in biocatalysis applications, and one example from is an ONIOM (MP2 HF) study of catechol O-methyltransferase [88],... 

Flucytosine is converted in Candida fungi to 5-fluorouracil by the action of a specific cytosine deaminase. As an antimetabolite, this compound disrupts DNA and RNA synthesis (p. 298), resulting in a fungicidal effect Given orally, flucytosine is rapidly absorbed. It is well tolerated and often combined with amphotericin B to allow dose reduction of the lattet... 

Huber BE, Austin EA, Richards CA, et al. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene Significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA 1994 91 8302-8306. 

Khil MS, Kim JH, Mullen CA, et al. Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene. Clin Cancer Res 1996 2 53-57. 

Ng EY, Rehemtulla A, Laurence TS, et al. Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine. Radiat Res Soc Proc 1977. 

L.D. Stegman, A. Rehemtulla, B. Beattie, E. Klevit, T.S. Lawrence, R.G. Blasberg, J.G. Tjuvajev, B.D. Ross, Noninvasive quantitation of cytosine deaminase transgene expression In human tumor xenografts with in vivo magnetic resonance spectroscopy, Proc. Natl. Acad. Scl. USA 96 (1999) 9821-9826. 

L.D. Stegman, A. Rehemtulla, D.A. Hamstra, D.J. Rice, S.J. Jonas, K.L. Stout, T.L. Chenevert, B.D. Ross, Diffusion MRI detects early events In the response of a glioma model to the yeast cytosine deaminase gene therapy strategy. Gene Then 7 (2000) 1005-1010. 

H. Corban-Wllhelm, W.E. Hull, G. Becker, U. Bauder-Wust, D. Greullch, J. Debus, Cytosine deaminase and thymidine kinase gene therapy In a dunning rat prostate... 

This field was recently reviewed [172]. Enzymes that have been used for ADEPT are, e.g., alkaline phosphatase, carboxypeptidase A, cytosine deaminase, -lactamase prodrugs and corresponding drugs were, e.g., adriamycin phosphate and adriamycin, methotrexate-alanine and methotrexate, 5-fluorocytosine and 5-fluorouracil, and vinca-cephalosporin and vinca alkaloid [172 and references therein]. 

Cunningham C, Nemunaitis J. A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer. Human Gene Ther 2001 12(12) 1594—1596. 

Examples of GDEPT include irinotecan (CPT-11), a prodrug of 7-ethyl- 10-hydroxy-camptothecin activated by carboxylesterase 5-fluorocytosine, a prodrug of 5-FU activated by cytosine deaminase and cyclophosphamide, a prodrug of 4-hydroxycyclophosphamide activated by cytochrome P450, which degrades into acrolein and phospho-ramide mustard.112-115... 

Brown, N. L., and Lemoine, N. R. Clinical trials with GDEPT Cytosine deaminase and 5-fluorocytosine. Methods Mol. Med. 90 451-458, 2004. 

Several other proteins show a low, but significant amino acid identity with atzA (Table 22.4). All of these, urease-alpha subunit (urea amidohydrolase), cytosine deaminase, and imidazolone-5-propionate hydrolase, catalyze hydrolytic reactions with substrates involved in the metabolism of nitrogenous compounds (Sadowsky et al 1998). A Rhizobium strain capable of atrazine dechlorination has been isolated from a soil that was previously treated with atrazine (Bouqard et al., 1997). This bacterium could not mineralize atrazine, and it accumulated hydroxyatrazine as the sole metabolite after long-term incubations. Interestingly, 22 of the 24 identified amino acids at the N-terminus of the atrazine halidohydrolase from Rhizobium were identical with atzA from Pseudomonas strain ADP. 

F. Kanai, K. H. Lan, Y. Shiratori, T. Tanaka, M. Ohashi, T. Okudaira, Y. Yo-shida, H. Wakimoto, H. Hamada, H. Nakabayashi, T. Tamaoki, and M. Omata, In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene, Cancer Res. 57 461 (1997). 

G. Cao, S. Kuriyama, J. Gao, M. Kikukawa, L. Cui, T. Nakatani, X. Zhang, H. Tsujinoue, X. Pan, H. Fukui, and Z. Qi, Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter, Gene Ther. 6 83 (1999). 

Harell RL, Rajanayagam S, Doanes AM, et al. Inhibition of vascular smooth muscle cell proliferation and neointimal accumulation by adenovirus-mediated gene transfer of cytosine deaminase, Circulation 1997 96 621-627. 

Examination of the crystals of yeast cytosine deaminase shows a second zinc ion coordinated to the active site by a bridging water/hydroxide ion to the catalytic zinc ion. This zinc ion has three other water molecules bound to it some of which have very short ( 1.8A) bonds to the second zinc. This may represent an inhibitory zinc ion that has bound to the protein with more than one hydroxide ion coordinated to the zinc. 

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