Adenine Deaminase Activity Research Articles

Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.

Ameliorative potentials of methanol leaf extract of Anthocleista vogelli on mercury chloride induced neurotoxicity

In this work, the effects of A. vogelli leaf extract on a few neurotoxicity indicators in Wistar rats were examined. Group 1 consisted of a normal control group, Group 2 of mercury (II) chloride, Group 3 of mercury (II) chloride + Diazepam 5 mg/kg, and Group 4 of mercury (II) chloride + plant extract 400 mg/kg bw. The sixteen (16) male rats were divided into these four groups at random. Animals were slaughtered and medications and extracts were administered for 21 days. Cerebellum and cerebrum brain samples were obtained and examined. Using accepted procedures, assays for acetylcholinesterase, alpha-tocopherol, and adenine deaminase were conducted. The acetylcholinesterase activity, vitamin E concentration, and adenine deaminase activity in the untreated groups were significantly (p≤0.05) higher than in the control group. Additionally, it was noted that both the plant extract and the usual medication, diazepam, showed significantly higher levels of vitamin E and adenine deaminase activity (p≤0.05) accompanied with lower acetycholinesterase activity. This suggests that the plant extract may have the potential to be a neuroprotective agent and could be used to treat neurotoxicity and other related disorders. Keywords: Neurotoxicity, acetylcholinesterase, alpha-tocopherol, adenine deaminase, Anthocleista vogelli.

Effects of iron supplementation on blood adenine deaminase activity and oxidative stress in Trypanosoma evansi infection of rats

The aim of this study was to assess the effects of iron supplementation on oxidative stress and on the activity of the adenosine deaminase (ADA) in rats experimentally infected by Trypanosoma evansi. For this purpose, 20 rats were divided into four experimental groups with five animals each as follows: groups A and B were composed by healthy animals, while animals from groups C and D were infected by T. evansi. Additionally, groups B and D received two subcutaneous doses of iron (60 mg kg-1) within an interval of 5 days. Blood samples were drawn on day 8 post infection in order to assess hematological and biochemical variables. Among the main results are: (1) animals from group C showed reduced erythrogram (with tendency to anemia); however the same results were not observed for group D; this might be a direct effect of free iron on trypanosomes which helped to reduce the parasitemia and the damage to erythrocytes caused by the infection; (2) iron supplementation was able to reduce NOx levels by inhibiting iNOS, and thus, providing an antioxidant action and, indirectly, reducing the ALT levels in groups Band D; (3) increase FRAP levels in group D; (4) reduce ADA activity in serum and erythrocytes in group C; however, this supplementation (5) increased the protein oxidation in groups B and D, as well as group C (positive control). Therefore, iron showed antioxidant and oxidant effects on animals that received supplementation; and it maintained the activity of E-ADA stable in infected/supplemented animals.

The catalase activity of diiron adenine deaminase

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

Phenotypic Consequences of Purine Nucleotide Imbalance inSaccharomyces cerevisiae

Coordinating homeostasis of multiple metabolites is a major task for living organisms, and complex interconversion pathways contribute to achieving the proper balance of metabolites. AMP deaminase (AMPD) is such an interconversion enzyme that allows IMP synthesis from AMP. In this article, we show that, under specific conditions, lack of AMPD activity impairs growth. Under these conditions, we found that the intracellular guanylic nucleotide pool was severely affected. In vivo studies of two AMPD homologs, Yjl070p and Ybr284p, indicate that these proteins have no detectable AMP, adenosine, or adenine deaminase activity; we show that overexpression of YJL070c instead mimics a loss of AMPD function. Expression of the yeast transcriptome was monitored in a AMPD-deficient mutant in a strain overexpressing YJL070c and in cells treated with the immunosuppressive drug mycophenolic acid, three conditions that lead to severe depletion of the guanylic nucleotide pool. These three conditions resulted in the up- or downregulation of multiple transcripts, 244 of which are common to at least two conditions and 71 to all three conditions. These transcriptome results, combined with specific mutant analysis, point to threonine metabolism as exquisitely sensitive to the purine nucleotide balance.

PCR vs. ADA for Diagnosing Tuberculous Pericarditis

Studies have suggested that either polymerase chain reaction (PCR) or adenine deaminase activity (ADA) may be more sensitive than direct microscopic

Potential markers of the nutritional status in an experimental model

The activity of adenine deaminase (ADA) and purine nucleoside phosphorylase (PNP) as potential nutritional markers was analyzed in an experimental model. Weanling Wistar rats were fed a protein-free diet ad libitum to obtain a severe degree of wasting. An age-matched control group received a stock diet. At the end of the experiment, body weight (BW) and thymus weight (TW) were determined. Activity of ADA and PNP was determined on thymocytes of protein-deprived and control rats; the results, expressed as micromoles of uric acid × 10 −1/W (W = TW/BW 0.75), were 17.0 ± 2.6 versus 9.1 ± 3.0 for ADA and 11.5 ± 4.2 versus 3.9 ± 1.0 for PNP ( P < 0.01). These results suggest that the nutritional stress provoked by the administration of a protein-free diet from weaning onward affects the development of thymocytes. Moreover, the increase in the activity of ADA and PNP would be an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which would be toxic for T lymphocytes. However, some investigators have observed an increase of ADA activity in human serum under some adverse conditions; for this reason and taking into account the present findings, it would be interesting to determine the relation between the actvity of ADA and PNP in thymocytes and serum in experimental models to analyze and propose these biochemical parameters as potential and useful markers of nutritional status; it also would be interesting to test this relation in human studies.

Catabolism of adenine nucleotides in suspension-cultured plant cells

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8-14C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14CO2. Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8-14C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest that participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.

A novel mechanism of mycophenolic acid resistance in the protozoan parasite tritrichomonas foetus

Tritrichomonas foetus relies primarily on the salvage of hypoxanthine to supply purine nucleotides. Mycophenolic acid disrupts T. foetus growth by specifically inhibiting inosine-5′-monophosphate (IMP) dehydrogenase, thereby blocking the biosynthesis of guanine nucleotides from hypo-xanthine. We have cloned a T. foetus strain (mpa r) that was 50-fold more resistant to mycophenolic acid than wild type ( ic 50 = 1 mM for mpa r vs 20μM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpa r was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpa r strains. Mycophenolic acid (100 μM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T. foetus mpa r, although no inhibition of T. foetus mpa r growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpa r so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T. foetus mpa r incorporated xanthine more efficiently into the nucleotide pool relative to hypoxanthine and guanine than wild type. Xanthine incorporation via XMP provided an IMP dehydrogenase independent route to guanine nucleotides that would enable the parasite to become mycophenolic acid resistant. No difference could be detected between wild type and mpa r hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpa r: it was deficient in hypoxanthine transport and had diminished adenine deaminase activity. The apparent net result of these two changes was to lower the intracellular concentration of hypoxanthine in mpa r. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpa r. The mpa r strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.

Enzymatic activities for interconversion of purines in spirochetes.

Enzymatic activities that catalyze the interconversion of purines and purine derivatives were detected in cell extracts of Spirochaeta aurantia, Spirochaeta stenostrepta, Treponema succinifaciens, and Treponema denticola. Phosphoribosyltransferase activities present in cell extracts of each of the four spirochete species functioned in the conversion of adenine, hypoxanthine, and guanine to AMP, IMP, and GMP, respectively. Nucleotidase activities in the extracts mediated the formation of nucleosides from nucleotides. The conversion of adenosine, inosine, and guanosine to the respective purine bases was catalyzed by nucleoside phosphorylase and, in some instances, by nucleoside hydrolase activities. Guanine deaminase activity was found in both S. aurantia and S. stenostrepta, whereas adenosine deaminase activity was detected only in S. aurantia. Adenine deaminase activity in T. succinifaciens extracts was sensitive to O2 and was relatively resistant to heating. Our results indicate that the four species of spirochetes studied possess a broad spectrum of purine interconversion enzymes. It is suggested that these enzymes may function in metabolic processes important for the survival of spirochetes in nutrient-poor natural environments.

Adenylate degradation in Escherichia coli. The role of AMP nucleosidase and properties of the purified enzyme.

Extracts of Escherichia coli K12 degrade AMP to hypoxanthine, adenine, adenosine, and inosine. Degradation experiments with mutants which lack purine nucleoside phosphorylase or both purine nucleoside phosphorylase and adenosine deaminase demonstrate that hypoxanthine formation is dependent on purine nucleoside phosphorylase. These findings are consistent with an absence of adenine deaminase activity in E. coli. Adenine is formed from AMP in extracts of the E. coli mutants as well as the wild type cells. This activity is due to AMP nucleosidase. Purified, homogeneous AMP nucleosidase gives a subunit Mr = 52,000 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 280,000 by comparative gel filtration. Kinetic studies with this enzyme give cooperative initial rate curves with AMP as substrate, with MgATP2- as an activator, and with Pi as an inhibitor. Phosphate inhibition is competitive with McATP2- (Ki = 0.2 mM) and reverses the activation by MgATP2-. In the absence of MgATP2-, the apparent S0.5 for AMP is 15 mM and decreases to 90 microM at saturating MgATP2-. The maximum rate of AMP hydrolysis is not affected by MgATP2-. Kinetics of MgATP2- activation give a constant for half-maximum activation varying from 120 microM in the presence of low AMP to approximately 2 microM when AMP is present at near saturation. Formycin 5'-PO4 is a powerful competitive inhibitor with respect to AMP, giving a Kis of 72 nM and a Km/Kis ratio of 1,200. Adenylate degradation experiments indicate that AMP nucleosidase is the major enzyme of AMP catabolism in E. coli. The kinetic properties of the purified enzyme indicate that regulation occurs by the intracellular MgATP2- /Pi ratio and the concentration of AMP.

Biochemical and electrophysiologic effects of thallium poisoning on the rat corpus striatum

Rats were given thallium acetate (5 mg of thallium/kg) ip daily for 7 days. The corpus striatum was removed, and several enzymes and protein estimations were made. Succinic dehydrogenase, monoamine oxidase, acid phosphatase, guanine deaminase and protease (cathepsin) activities in the corpus striatum were depleted by thallium, whereas adenosine triphosphatase and adenine deaminase activity were unaffected. Protein content of the corpus straitum was significantly increased, which could be due to a depletion of cathepsin and acid phosphatase; sulfhydryl-dependent enzymes were particularly depleted. Single neuron activity of the caudate nucleus, recorded after iv injection of thallium acetate (10 mg/kg body weigh), showed an increased firing rate of the caudate neurons, which could possibly be due to an imbalance of neurotransmitters.