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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD.
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PMID:Cytosine deaminase as a negative selective marker for Arabidopsis. 825 32

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

Gene therapy may allow targeted delivery of tumoricidal drugs to treat pancreatic cancer. Cytosine deaminase (CD) is a bacterial enzyme that converts the nontoxic agent 5-fluorocytosine (5FC) to the active chemotherapeutic agent 5-fluorouracil (5FU). Neoplastic cells induced to express the CD gene treated with 5FC may generate locally high concentrations of 5FU while minimising systemic toxicity. Replication deficient adenovirus vector carrying the CD gene (AdCMV.CD) was tested for therapeutic efficacy against the murine pancreatic carcinoma cell line Pan02. Pan02 cells were infected in vitro with AdCMV.CD or null vector (Ad.-Null) and were examined for expression of CD messenger RNA (mRNA) (Northern blot) and CD enzymatic function (spectrophotometry). mRNA transcripts of the CD gene increased in a dose-dependent manner after infection with AdCMV.CD. Conversion of 5FC to 5FU at a multiplicity of infection (MOI) of 20 was measured to be 51% after a 48-hr incubation. Growth inhibition was measured by MTT assay and thymidine uptake. Pan02 growth in vitro treated with AdCMV.CD and 5FC was inhibited by 80% as compared to cells treated with Ad.Null and 5FC. An in vivo model of pancreatic cancer was established by injecting 2.5 x 10(5) PAN02 cells subcutaneously into the flanks of C57BL/ 6 mice. Seven days later AdCMV.CD was injected into each tumor and 5FC was administered for 10 days. Treatment of mice with AdCMV.CD and 5FC inhibited tumor growth compared to mice who received AdCMV.CD only or 5FC only. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.
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PMID:In vivo adenoviral-mediated gene transfer in the treatment of pancreatic cancer. 920 75

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

Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcomes with transgene expression and will be useful in vector development. Cytosine deaminase (CD) is a microbial gene undergoing clinical trials in gene-directed enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversion of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-fluorouracil (5-FU). The feasibility of this approach is demonstrated in subcutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic fluxes of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodrug conversion (k(1)(app)), 5-FU efflux from the observable tumor volume (k(2)(app)), and formation of cytotoxic fluorinated nucleotides from 5-FU (k(3)(app)) were 0.49 +/- 0.27 min(-1), 0.766 +/- 0.006 min(-1), and 0.0023 +/- 0.0007 min(-1), respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above the in vitro K(m). These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expression in tumors. This capability provides an approach for measuring gene expression that will be useful in clinical gene therapy trials.
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PMID:Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy. 1044 78

Cytosine deaminase (CD) gene of E. coli converts the non-toxic compound 5-fluorocytosine (5-FC) into 5-fluorouracil. We have introduced a vector expressing the CD gene in a rat colon carcinoma cell line. Expression of the CD gene confers 5-FC sensitivity to these cells in vitro and in vivo. In a bifocal model consisting in a simultaneous engrafment of a CD+ tumor on one lobe of the liver and a wild-type parental tumor on the opposite lobe, treatment with 5-FC results in regression of both type of tumors, indicating the existence of a distant bystander effect.
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PMID:Regression of experimental liver tumor after distant intra-hepatic injection of cytosine deaminase-expressing tumor cells and 5-fluorocytosine treatment. 1067 68

Colon carcinoma accounts for 20% of deaths due to malignancies in the Western world. Once metastases occur, therapeutic options are limited, with an approximate 5-year survival of only 5%. To investigate the potential of new gene therapeutic approaches, a hepatic micrometastasis model of colon carcinoma in BALB/c mice was established. Inoculation of syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old mice resulted in the formation of multiple hepatic metastases. Selective transduction of developing hepatic metastases was demonstrated using a beta-galactosidase-expressing recombinant adenovirus. Cytosine deaminase (CD) can metabolize 5-fluorocytosine into the chemotherapeutic reagent 5-fluorouracil (5FU). The antitumoral potential of this suicide gene therapy approach was explored by systemic application of a recombinant replication-deficient adenovirus encoding for the bacterial CD gene under the control of the cytomegalovirus promoter (Ad.CMV-CD). Injection into the tail vein of tumor-bearing mice resulted in delayed tumor growth with significant reduction in hepatic metastases. The potential of this experimental approach for possible future clinical applications was evaluated by investigating adenoviral transduction efficiency, 5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a variety of human colon cancer cell lines. Although the murine cell lines MCA26 and CC36 were highly sensitive to 5FU, the human colon cancer cell lines showed a 1-100 times higher resistance to 5FU. Specific Ad.CMV-CD toxicity correlates with 5FU toxicity. Transduction efficiency in human colon carcinoma cell lines was shown to be 10-1700 times higher compared with murine cell lines, thus compensating for 5FU resistance. In conclusion, suicide gene therapy using CD may be promising as an adjuvant treatment regimen for hepatic micrometastases of human colon carcinoma.
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PMID:Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase. 1076 50

Cytosine deaminase (CD) is found in prokaryotes and fungi (but not higher eukaryotes) and catalyzes the deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. The former activity is an important function within the pyrimidine-salvage pathway, while the latter activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. Recombinant bacterial CD from Escherichia coli has been subcloned, overexpressed, purified and crystallized for structural analysis. The crystals belong to space group R32, with unit-cell parameters a = b = 109.1, c = 240 A and diffract to at least 1.5 A resolution at a synchrotron X-ray source. There is one enzyme subunit per asymmetric unit and the Matthews coefficient V(M) is 2.8 A(3) Da(-1), corresponding to a solvent content of 56%. Selenomethionine-containing protein has been prepared and crystallized for MAD phasing.
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PMID:Crystallization and preliminary X-ray analysis of bacterial cytosine deaminase. 1167 31

Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (alphabeta)(8) barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent.
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PMID:The structure of Escherichia coli cytosine deaminase. 1181 40


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