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 (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

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the Escherichia coli codA gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter psbDII. Cytosine deaminase converts 5'-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the psbDII promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the psbDII promoter will not express the codA gene and, consequently, will live on 5'-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the psbDII pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the codA gene to direct the timing of expression of codA to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.
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PMID:Genetic selection scheme for isolation of signal transduction pathway mutants. 1476 78

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

The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method. Cytosine deamination proceeds via a sequential mechanism involving the protonation of N(3), the nucleophilic attack of C(4) by the zinc-coordinated hydroxide, and the cleavage of the C(4)-N(4) bond. The last step is the rate determining step for the generation of the zinc bound uracil. Uracil is liberated from the Zn atom by an oxygen exchange mechanism that involves the formation of a gem-diol intermediate from the Zn bound uracil and a water molecule, the C(4)-O(Zn) cleavage, and the regeneration of the Zn-coordinated water. The rate determining step in the oxygen exchange is the formation of the gem-diol intermediate, which is also the rate determining step for the overall yCD-catalyzed deamination reaction.
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PMID:Catalytic mechanism of yeast cytosine deaminase: an ONIOM computational study. 1553 15

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

Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells (P < 0.006) and GFP-4T1 cells (P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.
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PMID:Osteoclasts direct bystander killing of cancer cells in vitro. 1613 79

Yeast cytosine deaminase, a zinc metalloenzyme, catalyzes the deamination of cytosine to uracil. Experimental and computational evidence indicates that the rate-limiting step is product release, instead of the chemical reaction step. In this work, we use molecular dynamics to suggest ligand exit paths. Simulation at 300 K shows that the active site is well protected by the C-terminal helix (residues 150-158) and F-114 loop (residues 111-117) and that on the molecular dynamics timescale water does not flow in or out of the active site. In contrast, simulation at 320 K shows a significant increase in flexibility of the C-terminal helix and F-114 loop. The motions of these two regions at 320 K open the active site and permit water molecules to diffuse into and out of the active site through two paths with one much more favored than the other. Cytosine is pushed out of the active site by a restraint method in two directions specified by these two paths. In path 1 the required motion of the protein is local-involving only the C-terminal helix and F-114 loop-and two residues, F-114 and I-156, are identified that have to be moved away to let cytosine out; whereas in path 2, the protein has to rearrange itself much more extensively, and the changes are also much larger compared to the path 1 simulation.
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PMID:A molecular dynamics study of the ligand release path in yeast cytosine deaminase. 1721 60

Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.
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PMID:Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo. 1948 91

Cytosine deaminase is a non-mammalian enzyme of widespread interest for prodrug enzyme therapy due to its ability to convert prodrug 5-fluorocytosine into anticancer drug 5-fluorouracil. Cytosine deaminase enzyme has been purified to homogeneity from E. coli K-12 MTCC 1302 strain. K(m) values for cytosine and 5-fluorocytosine were found to be 0.26 mM and 1.82 mM, respectively. We developed a chitosan-entrapped cytosine deaminase nanocomposite. Atomic force microscopy and transmission electron microscopy images showed an elongated sphere shape nanocomposite with an average size of 80 nm diameter. Fourier transform infrared spectroscopy and X-ray diffraction results confirmed gel formation and entrapment of cytosine deaminase within the nanocomposite. Sustained release of cytosine deaminase from the nanocomposite up to one week depicted its potential implication in prodrug inducted enzyme therapy.
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PMID:Synthesis and characterization of a novel chitosan based E. coli cytosine deaminase nanocomposite for potential application in prodrug enzyme therapy. 2096 Feb 22


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