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)

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.
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PMID:Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria. 16 27

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.
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PMID:Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea). 137 74

The activities of six bacteriophage T2r(+)-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 x 10(8) to 2 x 10(8) cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 x 10(8) to 8 x 10(8) per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.
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PMID:Relationship between Escherichia coli B titer and the level of deoxycytidylate deaminase activity induced on bacteriophage T2r + infection. 433 61

The biosynthesis of 2'-deoxyuridine monophosphate (dUMP) has been studied in a cytidine- and uracil-requiring mutant of Salmonella typhimurium (DP-55). The dUMP pool and the thymidine monophosphate (dTMP) pool of DP-55, grown in the presence of (3)H-uracil and unlabeled cytidine, are found to have the same specific activities. However, only 30% of the dUMP and the dTMP is synthesized from a uridine nucleotide. Seventy per cent is derived directly from a cytosine compound. The identification and partial purification of a Mg(2+)-dependent 2'-deoxycytidine triphosphate (dCTP) deaminase from S. typhimurium suggests that the combined action of dCTP deaminase and 2'-deoxyuridine triphosphate pyrophosphatase accounts for 70% of the dUMP, and therefore the dTMP, synthesized in vivo. The introduction of a thymine requirement (i.e., a block in thymidylate synthetase) into DP-55 results in a 100-fold increase in the size of the dUMP pool. However, the relative contribution of the uridine and cytidine pathways to dUMP synthesis is unaltered. The high dUMP pool is accompanied by extensive catabolism of dUMP to uracil. Partial thymine starvation of the cells results in a significant increase in the dUMP and dCTP pools. Moreover, an increase in the contribution of the dCTP pathway to dUMP synthesis is observed. As a result of these changes the catabolism of dUMP to uracil is augmented.
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PMID:Deoxycytidine triphosphate deaminase: identification and function in Salmonella typhimurium. 554 39

Methods are described for preparing and structurally analyzing two enzymes involved in the formation of dTMP, deoxycytidylate deaminase and thymidylate synthase. In the latter case, it has been possible through the use of recombinant DNA techniques with an amplification plasmid to obtain sufficient amounts of the E. coli and T4-phage synthases to complete the entire sequence of both enzymes by employing a combination of protein and DNA sequencing methods. A comparative analysis of the L. casei and E. coli synthases has revealed a 62% conservation of sequences but an even greater homology in their hydrophobic active site regions (82%), which are primarily hydrophobic in nature. The homology between these enzymes becomes apparent by deleting a 51 amino acid segment (residues 89-139) from the L. casei synthase, which accounts for the difference in size between these enzymes. Methods for obtaining the binding sites of both substrates are described, one being the activation of the carboxyls of folate with a water soluble carbodiimide and the other, the activation of dUMP by ultraviolet light. The DNA and protein sequence of the T4-phage synthase has recently been clarified by us and is in preparation. Of great interest is the finding by Purohit and Mathews (42), based on our sequence data for the synthase, that the gene segment for the carboxyl terminal end of dihydrofolate reductase overlaps with the amino end of the gene for thymidylate synthase. The complete amino acid sequence of T2-phage deoxycytidylate deaminase has been elucidated by conventional protein sequencing methods. The binding characteristics of this enzyme for its positive allosteric effectors and substrates, as determined by equilibrium dialysis, are consistent with the cooperative nature of its kinetic responses. Consistent with these findings was the demonstration that each of the enzyme's six subunits bound an equivalent amount of substrate or allosteric modifier. Similarly the deaminase showed a marked negative change in ellipticity at 280 nm in response to increasing concentrations of dCTP, changes which could be reversed by dTTP. From the information on the enzyme's primary sequence, it should be possible to define the substrate and allosteric binding regions within the deaminase with the appropriately activated compounds. A start in this direction has been initiated by the finding that dTTP is rapidly and apparently covalently fixed to the amino terminal cyanogen bromide peptide of the enzyme in the presence of ultraviolet light.
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PMID:Probing the infra-structure of thymidylate synthase and deoxycytidylate deaminase. 643 61

In view of the 20- to 80-fold elevation of deoxycytidine-5'-phosphate (dCMP) deaminase in many human malignant tumors, we have utilized 5-fluorodeoxycytidine ( FdCyd ) coadministered with tetrahydrouridine ( H4Urd ) as a combination of antitumor agents against two murine solid tumors which possess high levels of dCMP deaminase. This approach is based on our past studies in which we demonstrated that FdCyd is an excellent substrate for mammalian 2'-deoxycytidine kinase, and that H4Urd increases the toxicity of FdCyd in the mouse. Cell culture studies utilizing 2'- deoxytetrahydrouridine which inhibits cytidine deaminase and as 2'- deoxytetrahydrouridine -5'-monophosphate inhibits dCMP deaminase, provide indirect evidence for the pathway that we had proposed in the past, 2'- Deoxytetrahydrouridine antagonized the toxicity of FdCyd to a greater extent than did H4Urd and showed marked antagonism in cytidine deaminase-deficient cells. Cell lines lacking both cytidine and 2'-deoxycytidine-5'-monophosphate deaminase were markedly resistant to FdCyd . Thymidine and deoxyuridine antagonized toxicity in a manner consistent with the proposed pathway of anabolism of FdCyd and consistent with its resulting in the inhibition of thymidylate synthetase. We have established the efficacy of FdCyd + H4Urd chemotherapy utilizing adenocarcinoma 755 and Lewis lung carcinoma in C57BL X DBA/2 F1 mice. An example of an optimum schedule versus Lewis lung carcinoma is FdCyd , 10 to 12 mg/kg, plus H4Urd , 25 mg/kg, coadministered simultaneously, once per day on Days 1 to 7 after tumor implantation. Tumor inhibitions on Days 12, 14, and 16 were 95, 90, and 80%, respectively, with 8% maximum weight loss. Comparative studies were undertaken only with Lewis lung carcinoma and it was established that FdCyd + H4Urd surpasses the efficacies of 5-fluorouracil and 5-fluorodeoxyuridine as well as FdCyd when administered without H4Urd . We propose that the administration of FdCyd with H4Urd can result in preferential, tumor-directed conversion of a nontoxic nucleoside analogue to a toxic antimetabolite by an enzyme that is markedly elevated in human tumor tissue. The analogues of deoxycytidine are resistant to catabolism and are anabolized by a different subset of enzymes than are 5-fluorouracil or 5-fluorodeoxyuridine; therefore, it is a novel approach. Not only are there intrinsic selectivity, metabolic stability, and the advantages that accrue from prodrug therapy in this strategy, but in addition, the potential for an exclusively DNA-directed effect exists. This is in contrast to approaches with 5-fluorouracil and 5-fluorodeoxyuridine, in which, in addition to DNA effects, parallel effe
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PMID:Use of 5-fluorodeoxycytidine and tetrahydrouridine to exploit high levels of deoxycytidylate deaminase in tumors to achieve DNA- and target-directed therapies. 653 64

Recently, it has been shown, that 2-Chloro-deoxyadenosine (1), a series of analogues, and other DNA synthesis inhibitors, increased the deoxycytidine kinase (dCK) enzyme activity in different cells, without influencing thymidine kinase isoenzymes (TK1, TK2), dCMP-deaminase and thymidylate synthase (TS) activities (2,3). The dCK activity was 2-4 times higher in analogue treated cells, than in controls, which can not be explained by metabolic pool imbalance induced by the drugs. New mRNA and protein synthesis of dCK could not be detected, thus post-translational modification has been suggested for potentiation the activity of the dCK (1). Because secondary modifications of enzymes usually involve the signalling processes in cells, the universal G-protein activator fluorine ions were tested. dCK activity of human lymph node lymphocytes were increased 2-times, if cells were incubated in the presence of NaF for 1-2 hrs in cultures, while TK activity was not changed. The formation of dUTP from dCyd, was also enhanced by NaF, in parallel of dCK potentiation.
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PMID:Deoxycytidine kinase can be also potentiated by the G-protein activator NaF in cells. 959 3

We investigated directed deviations from the universal genetic code. Mutant tRNAs that incorporate cysteine at positions corresponding to the isoleucine AUU, AUC, and AUA and methionine AUG codons were introduced in Escherichia coli K12. Missense mutations at the cysteine catalytic site of thymidylate synthase were systematically crossed with synthetic suppressor tRNACys genes coexpressed from compatible plasmids. Strains harboring complementary codon/anticodon associations could be stably propagated as thymidine prototrophs. A plasmid-encoded tRNACys reading the codon AUA persisted for more than 500 generations in a strain requiring its suppressor activity for thymidylate biosynthesis, but was eliminated from a strain not requiring it. Cysteine miscoding at the codon AUA was also enforced in the active site of amidase, an enzyme found in Helicobacter pylori and not present in wild-type E. coli. Propagating the amidase missense mutation in E. coli with an aliphatic amide as nitrogen source required the overproduction of Cys-tRNA synthetase together with the complementary suppressor tRNACys. The toxicity of cysteine miscoding was low in all our strains. The small size and amphiphilic character of this amino acid may render it acceptable as a replacement at most protein positions and thus apt to overcome the steric and polar constraints that limit evolution of the genetic code.
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PMID:Reassigning cysteine in the genetic code of Escherichia coli. 975 88

Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure-function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42A and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77A and 2.10A, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase beta barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.
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PMID:Structural basis for recognition and catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jannaschii. 1290 16

2'-Deoxycytidylate deaminase [or deoxycytidine-5'-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5'-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5'-triphosphate (dCTP) as an activator and by deoxythymidine-5'-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 A. The protein forms a hexamer composed of subunits adopting a three-layer alpha/beta/alpha sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn(2+), a new conserved motif, (G(43)YNG(46)), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the gamma phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme-regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.
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PMID:Crystal structures of Streptococcus mutans 2'-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP x Mg2+. 1825 96


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