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Results for “Nucleotide Deaminases”

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At least 19 recordsLinked to original sources
PubMed1995

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.

Amino Acid Oxidoreductases↗
PubMed1991

Purification and properties of adenosine 5'-phosphosulfate deaminating enzyme from marine macroalga Gloiopeltis furcata.

An enzyme which catalyzed the hydrolytic removal of the 6-amino group of adenosine 5'-phosphosulfate (APS) into inosine 5'-phosphosulfate was purified from the marine red macroalga Gloiopeltis furcata by means of salt fractionation, affinity, anion-exchange, and hydrophobic interaction chromatographies. The native enzyme had a Mr of about 285,000. Dissociation yielded a form with a Mr of about 70,000. The enzyme catalyzed the irreversible deamination of adenosine and its 5'-substituted compounds in addition to APS. Thus the enzyme seemed to be a nonspecific adenine nucleotide deaminase. Some properties were determined and compared with those of other nonspecific adenine nucleotide deaminases.

Adenosine Phosphosulfate↗
PubMed1984

The distribution of adenosine deaminase, purine nucleotide phosphorylase and 5'-nucleotidase in subpopulations of thymocytes, bone marrow cells and other lymphoid organs in mice.

Lymphocyte populations of BALB/c mice were obtained from bone marrow, thymus, spleen, peripheral blood and lymphoid nodes. Subpopulations of thymocytes and bone marrow T-lymphocyte precursors were separated by density gradient centrifugation. The activity of adenosine deaminase (ADA) undergoes a marked increase during the evolution of bone marrow T-cell precursors to immature thymocytes, and a decrease with thymocytes maturation. The peripheral blood lymphocytes (PBL) present the lower activity of the enzyme, and lymphocytes from spleen (SL) and lymphoid nodes (LNL) show activity in the order of that in mature thymocytes. The activity of purine nucleotide phosphorylase (PNP) in the different lymphocytes populations experiments a very little variation with the T-lymphocyte differentiation. With the evolution of T-lymphocyte precursors to immature thymocytes the 5'-nucleotidase (5'-NT) activity experiment a 2-fold decrease. The thymocytes maturation is correlated with an increase in the activity of 5'-NT. The PBL present the maximal activity of the enzyme, whereas in spleen and LNL its levels of activity are in the range of that in mature thymocytes and bone marrow T-cell precursors respectively.

5'-Nucleotidase↗
PubMed2004

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes.

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

Alleles↗
PubMed2005

Crystal structure of a bifunctional deaminase and reductase from Bacillus subtilis involved in riboflavin biosynthesis.

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.

Amino Acid Sequence↗
PubMed1996

RNA editing: how a message is changed.

Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.

Animals↗
PubMed2006

The nucleotide targets of somatic mutation and the role of selection in immunoglobulin heavy chains of a teleost fish.

Sequence analysis of H chain cDNA derived from the spleen of an individual catfish has shown that somatic mutation occurs within both the VH- and JH-encoded regions. Somatic mutation preferentially targets G and C nucleotides with approximately balanced frequencies, resulting in the predominant accumulation of G-to-A and C-to-T substitutions that parallel the activation-induced cytidine deaminase nucleotide exchanges known in mammals. The overall mutation rate of A nucleotides is not significantly different from that expected by sequence-insensitive mutations, and a significant bias exists against mutations occurring in T. Targeting of mutations is dependent upon the sequence of neighboring nucleotides, allowing statistically significant hotspot motifs to be identified. Dinucleotide, trinucleotide, and RGYW analyses showed that mutational targets in catfish are restricted when compared with the spectrum of targets known in mammals. The preferential targets for G and C mutation are the central GC positions in both AGCT and AGCA. The WA motif, recognized as a mammalian hotspot for A mutations, was not a significant target for catfish mutations. The only significant target for A mutations was the terminal position in AGCA. Lastly, comparisons of mutations located in framework region and CDR codons coupled with multinomial distribution studies found no substantial evidence in either independent or clonally related VDJ rearrangements to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.

Amino Acid Motifs↗
PubMed1992

Cloning and nucleotide sequence of the cDNA encoding human erythrocyte-specific AMP deaminase.

The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5'-AATAAA) located at 1212 bp 3' downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.

AMP Deaminase↗
PubMed1983

The influence of the nucleus on the regulation of the dCMP deaminase in acetabularia.

The enzyme activity of dCMP deaminase (EC 3.5.4.12) In Acetabularia mediterranea is substantially increased at the beginning of cyst formation. A similar albeit advanced increase is observed in anucleate cells. The advance in the anucleate cells is not due to non-specific effects of cell surgery since nucleate grafts which are also subjected to surgery exhibit a delayed regulation characteristic for nucleate cells. Moreover, the nuclear effect on the regulation is demonstrated by experiments in which the flow of information from nucleus to cytoplasm is interrupted by treatment with actinomycin D or by tying off the rhizoid for five days. In these cells not only is the time of the increase advanced but also cyst formation is accelerated.

Cell Nucleus↗
PubMed1998

Single-cell measurements of purine release using a micromachined electroanalytical sensor.

To study the cellular events surrounding the formation of purines in cardiac ischemia, we have micromachined a micrometer-scale titer chamber containing an integrated electrochemical sensor, capable of measuring analytes produced by a single heart cell. The analytical procedure involves the determination of metabolites via the amperometric detection of enzymically generated hydrogen peroxide, measured at a platinized microelectrode, poised at a suitably oxidizing potential, equivalent to +420 mV vs Ag/AgCl. Signals were recorded as current-time responses and were integrated to give a total charge (Q) attributable to the reaction under investigation. The amount of analyte produced by the cell was subsequently quantified by the addition of a known amount of calibrant. As a consequence, by using a cascade of three enzymes (adenosine deaminase, nucleotide phosphorylase, and xanthine oxidase), we were able to show that, after rigor contracture had been induced in a single myocyte, adenosine (but not inosine) only reached the extracellular space after the cell membrane had been permeabilized by detergent. These data, which could only be obtained unambiguously by using this single-cell methodology, have provided us with information on the origin of ischemic adenosine which challenges the established assumption that purine release is an early retaliatory response from intact anoxic myocytes.

Biosensing Techniques↗
PubMed1994

5-Aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase from human CCRF-CEM leukemia cells: purification, pH dependence, and inhibitors.

The bifunctional enzyme 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase-IMP cyclohydrolase has been purified 780-fold to apparent homogeneity from human CCRF-CEM leukemia cells, completed with chromatography on Affi-Gel Blue followed by AICAR-Sepharose 4B. Using a sensitive radioassay, IMP cyclohydrolase has a Ks value for 5-formamidoimidazole-4-carboxamide ribotide (FAICAR) at pH 7.4 of 0.87 +/- 0.11 microM. The following purine nucleotide derivatives were potent competitive inhibitors of IMP cyclohydrolase: 2-mercaptoinosine 5'-monophosphate (Ki = 0.094 +/- 0.024 microM), xanthosine 5'-monophosphate (Ki = 0.12 +/- 0.01 microM), 2-fluoroadenine arabinoside 5'-monophosphate (Ki = 0.16 +/- 0.02 microM), 6-mercaptopurine riboside 5'-monophosphate (Ki = 0.20 +/- 0.02 microM), adenosine N1-oxide 5'-monophosphate (Ki = 0.28 +/- 0.03 microM), and N6-(carboxymethyl)adenosine 5'-monophosphate (Ki = 1.7 +/- 0.42 microM). The pH dependencies of Vmax and Vmax/Ks values for IMP cyclohydrolase are consistent with a single ionizable amino acid residue (pKa = 7.57 +/- 0.09) of the enzyme which must be unprotonated for catalysis to occur and a residue (pKa = 7.57 +/- 0.14) which must be unprotonated for FAICAR to bind. The pKa values of 5.81 +/- 0.03 and 9.41 +/- 0.04 determined for FAICAR indicate that ionization of the substrate does not contribute significantly to the pH effects observed. Chemical modification of IMP cyclohydrolase provides evidence for arginine and cysteine residues at the active site, and roles for these residues in the mechanism of catalysis are proposed.

Acyltransferases↗