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)

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.
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PMID:Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine. 1091 4

The presence of adenine in the L-alanine defined medium substantially inhibited the growth of the moderately halophilic eubacterium Halomonas elongata. Extensive attempts to reverse the adenine toxicity for growth were made using a variety of purine and pyrimidine compounds, vitamins, and amino acids. Of the compounds tested, only cytosine was found to reverse the adenine growth inhibition. This indicates a mechanism similar to that found for some strains of Escherichia coli in which the presence of exogenous purines (e.g. adenine) was found to stop purine de novo synthesis and repress the synthesis of the pyrimidine salvage enzyme cytosine deaminase. H. elongata was found to possess an active adenine uptake system that was sodium dependent with only lithium having a considerable capacity to replace the sodium. A competition study indicated that the adenine transport system was quite specific. This paper represents the initial study of purine and pyrimidine salvage pathways and adenine uptake for the moderately halophilic eubacteria.
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PMID:Adenine toxicity and transport in the moderately halophilic eubacterium Halomonas elongata. 1144 64

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.
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PMID:Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers. 1224 48

Cytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively. The enzyme forms an alpha/beta fold similar to bacterial cytidine deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates.
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PMID:The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy. 1290 27

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.
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PMID:Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference. 1524 53

Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.
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PMID:Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy. 1538 61

Primary resistance in Candida albicans to flucytosine (5-FC) was investigated in 25 strains by identifying and sequencing the genes FCA1, FUR1, FCY21, and FCY22, which code for cytosine deaminase, uracil phosphoribosyltransferase (UPRT), and two purine-cytosine permeases, respectively. These proteins are involved in pyrimidine salvage and 5-FC metabolism. An association between a polymorphic nucleotide and resistance to 5-FC was found within FUR1 where the substitution of cytidylate for thymidylate at nucleotide position 301 results in the replacement of arginine with cysteine at amino acid position 101 in UPRT. Isolates that are homozygous for this mutation display increased levels of resistance to 5-FC, whereas heterozygous isolates have reduced susceptibility. Three-dimensional protein modeling of UPRT suggests that the Arg101Cys mutation disturbs the quaternary structure of the enzyme, which is postulated to compromise optimal enzyme activity. A single resistant isolate, lacking the above polymorphism in FUR1, has a homozygous polymorphism in FCA1 that results in a glycine-to-aspartate substitution at position 28 in cytosine deaminase.
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PMID:Molecular mechanisms of primary resistance to flucytosine in Candida albicans. 1550 67

Using 5-fluoropyrimidine analogues, high-performance liquid chromatography (HPLC), and the feeding of pyrimidine compounds to pyrimidine auxotrophs, the pathways for salvage of exogenous pyrimidine nucleosides and bases in Streptomyces were established. Selection for resistance to the analogues resulted in the isolation of strains of S. griseus lacking the following enzyme activities: uracil phosphoribosyltransferase (upp) and cytidine deaminase (cdd). The conversion of substrates in the pathway was followed using reverse-phase HPLC. The strains deficient in salvage enzymes were also verified by this method. In addition, feeding of exogenous pyrimidines to strains lacking the biosynthetic pathway confirmed the salvage pathway. Data from the analogue, HPLC, and feeding experiments showed that Streptomyces recycles the pyrimidine base uracil, as well as the nucleosides uridine and cytidine. Cytosine is not recycled due to a lack of cytosine deaminase.
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PMID:Pathways of pyrimidine salvage in Streptomyces. 1569 58

We describe positive/negative selectable cytosine deaminase MX cassettes for use in Saccharomyces cerevisiae. The basis of positive selection for cytosine deaminase (Fcy1) activity is that (a) fcy1 strains are unable to grow on medium containing cytosine as a sole nitrogen source and (b) fcy1 ura3 strains are unable to grow on medium containing cytosine as the sole pyrimidine source. Conversely, as 5-fluorocytosine (5FC) is toxic to cytosine deaminase-producing cells, fcy1 strains are resistant to 5FC. FCY1MX and FCA1MX cassettes, containing open reading frames (ORFs) of S. cerevisiae FCY1 and Candida albicans FCA1, respectively, were constructed and used to disrupt targeted genes in S. cerevisiae fcy1 strains. In addition, new direct repeat cassettes, kanPR, FCA1PR, FCY1PR and CaURA3PR, were developed to allow efficient deletion of target genes in cells containing MX3 repeats. Finally, the FCY1- and FCA1MX3 or PR direct repeat cassettes can be readily recycled after 5FC counter-selection on both synthetic and rich media.
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PMID:Cytosine deaminase MX cassettes as positive/negative selectable markers in Saccharomyces cerevisiae. 1608 73

The success of cancer gene therapies requiring in vivo gene transfer is severely hampered by the low efficacy of gene transfer, which has been difficult to improve. We therefore established a novel strategy to increase the share of transduced cells post gene transfer. We hypothesized that in vivo selection of tumor cells transduced with a suicide gene effectively enriches these cells within a tumor, thus allowing for an increased bystander effect after the prodrug is given, leading to enhanced eradication of tumor cells. We reasoned that in vivo enrichment should be achieved by exploiting the metabolism of the suicide gene product. For this 'enrichment-eradication' strategy we chose a fusion gene of cytosine deaminase and uracil phosphoribosyl transferase. Positive selection (enrichment) was to be achieved by concurrently giving N-(phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine de novo synthesis, which leads to pyrimidine depletion-mediated death of non-transduced cells, and cytosine, to rescue fusion gene expressing cells via the pyrimidine salvage pathway. Negative selection (eradication) was to be induced by giving the prodrug 5-fluorocytosine. Indeed, murine NXS2 neuroblastoma cells transduced with the fusion gene were effectively enriched in vitro, leading to a near-complete bystander effect. In vivo enrichment-eradication of NXS2 cells led to decreased tumor growth. This proof-of-principle study shows that enrichment-eradication may compensate the effects of low in vivo gene transfer efficacy, a major obstacle in cancer gene therapy.
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PMID:Enriching suicide gene bearing tumor cells for an increased bystander effect. 1702 30


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