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)

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

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

Plasmodium knowlesi provides a highly versatile transfection system for malaria, since it enables rapid genetic modification of the parasite both in vivo as well as in vitro. However, it is not possible to perform multiple genetic manipulations within one parasite line because of a lack of selectable markers. In an effort to develop additional selectable markers for this parasite, positive and negative selectable markers that have recently been successfully used in Plasmodium falciparum were tested. It was shown that the positive selectable markers human dihydrofolate reductase (hdhfr), blasticidin S deaminase (bsd) and neomycin phosphotransferase II (neo) all conferred drug resistance to P. knowlesi when introduced as episomes. The plasmid containing the hdhfr selectable marker was not only successfully introduced as circular form, but also as linear fragment, demonstrating for the first time single crossover integration in P. knowlesi. Thymidine kinase was tested for its potential as negative selectable marker and it was shown that recombinant P. knowlesi parasites expressing thymidine kinase from episomes were highly sensitive to ganciclovir compared to wild-type P. knowlesi. The availability of new positive selectable markers and a strong candidate for a negative selectable marker for P. knowlesi, in combination with the opportunity to perform targeted single crossover integration in P. knowlesi, significantly increases the flexibility of this transfection system, making it one of the most versatile systems available for Plasmodium.
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PMID:New selectable markers and single crossover integration for the highly versatile Plasmodium knowlesi transfection system. 1474 47

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-A resolution displayed a tetrameric ring-like structure with an extensive interface of approximately 2400 A(2)/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.
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PMID:Crystal structure of a bifunctional deaminase and reductase from Bacillus subtilis involved in riboflavin biosynthesis. 1630 16

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.
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PMID:The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism. 1776 62

Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu51 and Lys79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-A resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu290, instead of the previously proposed Asp199, would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.
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PMID:Complex structure of Bacillus subtilis RibG: the reduction mechanism during riboflavin biosynthesis. 1898 85