Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.
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PMID:Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of the glnLG and purR genes in the regulation of codA expression. 267 19

Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.
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PMID:Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F. 314 44

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogeneous. The molecular weight of cytosine deaminase was determined to be approx. 230000 containing four identical subunits with each subunit having a molecular weight 54000. Cytosine was deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50 degrees C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The Km and V values for cytosine were 0.74 mM and 47.16 mumole/min, respectively. As effectors of enzyme activity, PPi stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171-1174).
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PMID:Purification and some properties of cytosine deaminase from Salmonella typhimurium. 675 59

Current treatments for metastatic malignant disease are often ineffective. One of the most promising of the selective genetic strategies against cancer is VDEPT (virally directed enzyme prodrug therapy). This uses a viral vector to carry a prodrug-activating enzyme gene into both tumour and normal cells. By linking the foreign gene downstream of tumour-specific transcription units, tumour-specific expression of the foreign enzyme gene can be achieved. We have developed a genetic therapy strategy using VDEPT against cancers that overexpress the oncogene ERBB2. This occurs in approximately one-third of breast and pancreatic tumours (and in a smaller proportion of other tumours) and involves transcriptional up-regulation of the ERBB2 gene with or without gene amplification. We have constructed a chimeric minigene consisting of the proximal ERBB2 promoter linked to the gene encoding cytosine deaminase, an enzyme that can deaminate the prodrug 5-fluorocytosine (5-FC) to form cytotoxic 5-fluorouracil (5-FU). We have constructed a double-copy recombinant retrovirus to deliver the enzyme gene under the control of the ERBB2 promoter into a panel of ERBB2 expression-positive (ERBB2+) and -negative (ERBB2-) pancreatic and breast cell lines. Cytosine deaminase activity was high in ERBB2+ transduced cells but was not detected in ERBB2- transduced cells. Significant cell death was observed in ERBB2+ transduced cells treated with 5-FC whereas ERBB2- cells were not affected. Hence we present a novel gene therapy strategy that is potentially tumour-specific and could be used against a range of tumour types that overexpress the ERBB2 oncogene.
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PMID:Gene therapy for cancer using tumour-specific prodrug activation. 758 78

Cytosine deaminase (CDase, EC 3.5.4.1) isolated from Escherichia coli contains a catalytically essential divalent metal ion. Fe2+ was efficiently removed from the enzyme with o-phenanthroline to yield an apoenzyme with less than 5% of the catalytic activity of native enzyme. The time courses for inactivation and for removal of Fe2+ from the enzyme by o-phenanthroline were similar. Apoenzyme reconstituted with Fe2+, Mn2+, Co2+, or Zn2+ (M2+CDase) had kcat values of 185, 88, 50, and 32 s-1, respectively. The Km values of these M2+CDases for cytosine were similar (0.22-0.39 mM). Cytosine potently inhibited reconstitution of the apoenzyme with Fe2+. Fe2+CDase was rapidly inactivated by 1 mM H2O2 (t1/2 < 1 s), whereas Mn2+CDase, Co2+CDase, and Zn2+CDase were not inactivated by H2O2. CDase was also inhibited by excess divalent cations. Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhibition constants of 1.8 and 5.8 microM, respectively. Cu2+ dissociated slowly from the secondary binding on CDase with a rate constant of 2 x 10(-3) s-1.
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PMID:Cytosine deaminase. The roles of divalent metal ions in catalysis. 822 44

Cytosine deaminase (EC 3.5.4.1), a non-mammalian enzyme, catalyzes the deamination of cytosine and 5-fluorocytosine to form uracil and 5-fluorouracil, respectively. Eukaryotic cells have been genetically modified with a bacterial cytosine deaminase gene to express a functional enzyme. When the genetically modified cells are combined with 5-fluorocytosine, it creates a potent negative selection system, which may have important applications in cancer gene therapy. In this paper, we introduce a novel positive selection method based upon the expression of the cytosine deaminase gene. This method utilizes inhibitors in the pyrimidine de novo synthesis pathway to create a condition in which cells are dependent on the conversion of pyrimidine supplements to uracil by cytosine deaminase. Thus, only cells expressing the cytosine deaminase gene can be rescued in a positive selection medium.
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PMID:Cytosine deaminase gene as a positive selection marker. 863 98

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.
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PMID:Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae. 951 58

To study the possibility of oral gene therapy using live attenuated Salmonella, eukaryotic expression vectors EGFPN1, pLCDSN were introduced into a live attenuated AraA(-) auxotrophic mutant of Salmonella typhimurium (SL3261) and were administered orally to BALB/c and C57BL/6 mice. After six weeks, these mice were challenged with 4T(1) and Lewis cancer cells. Until the tumors reached to about 10 mm in diameter, 5-fluorocytosine was given through intraperitoneal injection. Flow cytometry, confocal microscopy and PCR methods were used to detect the integration and expression of the genes. The inhibition of the tumor and the survival time of the mice were also investigated. Results showed that cytosine deaminase gene integration could be detected in almost all kinds of mice tissue. And the GFP expression was much stronger in spleen and tumor than in other tissues. Cytosine deaminase/5-fluorocytosine system had significant antitumoractivities in vivo. The anti-tumor activities of cytosine deaminase/5-fluorocytosine at 500 mg/kg on 4T(1) and Lewis carcinoma in BALB/c and C57BL/6 mice were more potent than the efficiency of 5-fluorouracil 10 mg/kg(P 0.05). Therefor, this experiment demonstrates the potential value of live attenuated Salmonella as carrier for oral gene therapy.
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PMID:Treatment of Tumor in Mice by Oral Administration of Cytosine Deaminase Gene Carried in Live Attenuated Salmonella. 1205 Aug 17

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.
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PMID:Nitrogen regulation of the codBA (cytosine deaminase) operon from Escherichia coli by the nitrogen assimilation control protein, NAC. 1270 Feb 71

Cytosine deaminase is an attractive candidate for anticancer gene therapy through its catalysis of the deamination of the prodrug 5-fluorocytosine to 5-fluorouracil. Recombinant yeast cytosine deaminase has been crystallized with the inhibitor 2-hydroxypyrimidine in 10% 2-propanol, 20% polyethylene glycol 4000, 0.1 M HEPES pH 7.5. The crystals belong to space group P2(1), with unit-cell parameters a = 45.31, b = 53.33, c = 64.29 A, beta = 99.98 degrees and one dimer per asymmetric unit. The crystals diffract X-rays beyond 1.5 A resolution and an initial atomic model has been built based on selenomethionyl multiwavelength anomalous data at 2 A resolution.
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PMID:Crystallization and preliminary crystallographic analysis of yeast cytosine deaminase. 1277 21


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