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The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis. 相似文献
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Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional delta hgprt/delta xprt mutant strain that exhibits an absolute requirement for 2'-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the delta hgprt/delta xprt strain cannot proliferate indefinitely without 2'-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The delta hgprt/delta xprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates. 相似文献
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Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana 总被引:1,自引:1,他引:0
Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH4 )2 SO4 precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)-1 min-1 . Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn2+ or Mg2+ ). In the presence of MnCl2+ other divalent cations (Mg2+ , Ca2+ , Ba2+ , Hg2+ and Cd2+ ) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The Km for adenine was 4.5±1.5 μ M , the Km for PRPP was 0.29±0.06 m M and the Ki for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The Km for benzyladenine was approximately 0.73±0.06 m M 相似文献
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Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells 总被引:4,自引:3,他引:1
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A. W. Murray 《The Biochemical journal》1967,103(1):271-279
1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme. 相似文献
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Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme 总被引:4,自引:0,他引:4
J M Wilson T E O'Toole P Argos D S Shewach P E Daddona W N Kelley 《The Journal of biological chemistry》1986,261(29):13677-13683
We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme. 相似文献
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The bifunctional flavoenzyme 5-hydroxyvaleryl-CoA dehydratase/ dehydrogenase has been crystallized from solutions containing ammonium sulfate (form I) or polyethylene glycol (form II) as precipitant. In both cases, the crystals grew in the monoclinic space group C2. The unit cell dimensions for form I crystals were determined as a = 162.8 Å, b = 71.8 Å, c = 83.5 Å, β = 109.1°. Corresponding values for form II crystals were a = 161.2 Å, b = 71.6 Å, c = 82.2 Å, β = 109.3°. In both cases most probably there are two monomers per asymmetric unit. The crystals diffract to about 2 Å resolution and are rather stable in the X-ray beam. © 1994 Wiley-Liss, Inc. 相似文献
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Human adenine phosphoribosyltransferase: Characterization from subjects with a deficiency of enzyme activity 总被引:1,自引:0,他引:1
Timothy E. O'Toole James M. Wilson M. Henry Gault William N. Kelley 《Biochemical genetics》1983,21(11-12):1121-1134
Adenine phosphoribosyltransferase (APRT) was characterized with respect to specific activity and immunoreactive protein (CRM) levels in hemolysate from 18 members of an APRT-deficient kindred. In addition, lymphoblastoid cell lines were established from six of these subjects and APRT from these cells was characterized in a similar fashion. Levels of specific activity and CRM in patients homozygous for the deficiency were less than 1% of normal. Heterozygous subjects had higher levels of activity and CRM in lymphoblasts than in erythrocytes and, in all cases, the APRT present was normal in terms of isoelectric point, subunit molecular weight, and heart stability. The higher levels of activity and CRM found in lymphoblasts may be due either to expression of a mutant gene product stabilized in a normal:mutant dimer or to autologous regulation. 相似文献
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We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed. 相似文献
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Y Moriwaki T Yamamoto J Yamakita S Takahashi Z Tsutsumi K Higashino 《Biochimica et biophysica acta》1999,1427(3):385-391
To determine whether interferon-gamma affects rat purine catabolic and salvage enzyme activities, rats were injected with interferon-gamma (600000 U/kg, i.p.) and, similarly to a vehicle-injected control group, killed before or after injection at 6, 12, and 24 h. Organ homogenates were prepared and enzymatic reactions with substrates were carried out, after which the products were measured either chromatographically or spectrophotometrically. Western and Northern blotting also were performed. In contrast to the vehicle-injected rats, interferon-gamma-injected rats showed a significant rise in xanthine oxidoreductase activity in the liver, while enzyme activity was unchanged in the spleen, kidney, and lung. Western analysis of hepatic xanthine oxidoreductase showed an increased concentration of this protein 12 and 24 h after interferon-gamma injection. Northern analysis disclosed an enhanced mRNA expression coding for this enzyme, peaking 12 h after injection. Contrastingly, the activities of adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase were not affected by interferon-gamma in any organ tested. While interferon-gamma causes an increased hepatic biosynthesis of xanthine oxidoreductase, the physiologic role of this enzyme induction remains undetermined. 相似文献
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Boitz JM Strasser R Hartman CU Jardim A Ullman B 《The Journal of biological chemistry》2012,287(10):7626-7639
Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K(m) of 15.4 μM, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani. 相似文献
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Cloning of a Drosophila melanogaster adenine phosphoribosyltransferase structural gene and deduced amino acid sequence of the enzyme 总被引:4,自引:0,他引:4
The Aprt locus of Drosophila melanogaster encodes the structural gene for adenine phosphoribosyltransferase (APRT). DNA cloned from microdissected salivary gland polytene chromosome region 62B7-12 was used in conjunction with chromosome walking and hybrid selection of mRNA to isolate the Aprt gene. Aprt lies at cytogenetic position 62B9 and is closely flanked by other genes of unknown function. Nucleotide sequencing shows that four APRT cDNAs have a common 5' terminus with an apparent cap consensus sequence but two different 3' sites of polyadenylation. The distribution of conserved amino acid sequences in APRT from vertebrates, insects and bacteria suggests that they may have shared a common ancestral gene for this ubiquitous enzyme. 相似文献
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The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P41212 or the enantiomorph P43212, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc. 相似文献
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Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis. 总被引:4,自引:1,他引:3
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The isolation of mutants defective in adenine metabolism in Bacillus subtilis has provided a tool that has made it possible to investigate the role of adenine deaminase in adenine metabolism in growing cells. Adenine deaminase is the only enzyme that can deaminate adenine compounds in B. subtilis, a reaction which is important for adenine utilization as a purine and also as a nitrogen source. The uptake of adenine is strictly coupled to its further metabolism. Salvaging of adenine is inhibited by the stringent response to amino acid starvation, while the deamination of adenine is not. The level of adenine deaminase was reduced when exogenous guanosine served as the purine source and when glutamine served as the nitrogen source. The enzyme level was essentially the same whether ammonia or purines served as the nitrogen source. Reduced levels were seen on poor carbon sources. The ade gene was cloned, and the nucleotide sequence and mRNA analyses revealed a single-gene operon encoding a 65-kDa protein. By transductional crosses, we have located the ade gene to 130 degrees on the chromosomal map. 相似文献
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