Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution

The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.     

Enzymatic synthesis of 2'-deoxyguanosine with nucleoside deoxyribosyltransferase-II

Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2'-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2'-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.     

Deoxyribosyl transfer catalysis with trans-N-deoxyribosylase. Kinetic study of purine(pyrimidine) to pyrimidine(purine) trans-N-deoxyribosylase.

Kinetic studies were carried out in order to investigate the enzymic mechanism of a 215-fold-purified purine(pyrimidine) nucleoside: purine(pyrimidine) deoxyribosyl transferase fraction from Lactobacillus helveticus. A variety of natural deoxyribonucleosides and bases were used as substrates. Initial velocity, product inhibition and isotopic exchange studies are consistent with a ping-pong bi-bi mechanism. The kinetic parameters are used to show that this fraction is free from any contamination by a specific purine nucleoside: purine deoxyribosyl transferase also found in the same strain of L. helveticus.     

The control of cell proliferation by preformed purines: a genetic study. I. Isolation and preliminary characterization of Chinese hamster lines with single or multiple defects in purine "salvage" pathways.

Sublines with single or multiple defects in purine "salvage" enzymes were isolated from the Chinese hamster fibroblastic line GMA32 through single or successive one-step selections for resistance to purine analogs. They were examined for their ability to incorporate purine bases and nucleosides into macromolecules, for their sensitivity to growth inhibitory purines, and for their rescue by exogenous purines from deprivation imposed by metabolic inhibitors of endogenous synthesis. The results show that a deficiency of either adenosine kinase (EC 2.7.1.20), adenine phosphoribosyltransferase (EC 2.4.2.7) or hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) abolishes the ability of adenine to cause cell death by interfering with pyrimidine synthesis; on the other hand, the pyrimidine starvation caused by adenosine is fully prevented only by a deficiency of adenosine kinase.     

Docking simulation with a purine nucleoside specific homology model of deoxycytidine kinase, a target enzyme for anticancer and antiviral therapy

5'-Phosphorylation, catalyzed by human deoxycytidine kinase (dCK), is a crucial step in the metabolic activation of anticancer and antiviral nucleoside antimetabolites, such as cytarabine (AraC), gemcitabine, cladribine (CdA), and lamivudine. Recently, crystal structures of dCK (dCKc) with various pyrimidine nucleosides as substrates have been reported. However, there is no crystal structure of dCK with a bound purine nucleoside, although purines are good substrates for dCK. We have developed a model of dCK (dCKm) specific for purine nucleosides based on the crystal structure of purine nucleoside bound deoxyguanosine kinase (dGKc) as the template. dCKm is essential for computer aided molecular design (CAMD) of novel anticancer and antiviral drugs that are based on purine nucleosides since these did not bind to dCKc in our docking experiments. The active site of dCKm was larger than that of dCKc and the amino acid (aa) residues of dCKm and dCKc, in particular Y86, Q97, D133, R104, R128, and E197, were not in identical positions. Comparative docking simulations of deoxycytidine (dC), cytidine (Cyd), AraC, CdA, deoxyadenosine (dA), and deoxyguanosine (dG) with dCKm and dCKc were carried out using the FlexX docking program. Only dC (pyrimidine nucleoside) docked into the active site of dCKc but not the purine nucleosides dG and dA. As expected, the active site of dCKm appeared to be more adapted to bind purine nucleosides than the pyrimidine nucleosides. While water molecules were essential for docking experiments using dCKc, the absence of water molecules in dCKm did not affect the ability to correctly dock various purine nucleosides.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine-xanthine-guanine phosphoribosyltransferase produced by this organism.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

Determination of ribonucleosides,deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.     

Purine metabolism by intracellular Chlamydia psittaci.

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.     

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.     

Further characterization of a depurinated DNA-purine base insertion activity from cultured human fibroblasts.

The purification from cultured human fibroblasts of a protein that binds specifically to partially depurinated DNA and inserts purines into those sites is described. The purine insertion, but not the binding, requires K+. The DNA binding can be saturated with increasing apurinic sites and is weakened by the presence of adenine or guanine. Base insertion into depurinated DNA is specific for adenine or guanine; none is observed with dATP or dGTP. When the depurinated DNA substrate is specifically cleaved with apurinic endonuclease, no purine insertion occurs. Guanine insertion does not occur into tRNA or depyrimidinated DNA, and thymine is not inserted into either depyrimidinated DNA or depurinated DNA. Purine insertion activity follows Michaelis-Menten kinetics with respect to purintes; the apparent Km values for both adenine and guanine are 5 microM. The enzyme binds the purine bases very tightly. Adenine binding saturates at less than 1 microM adenine, perhaps reflecting the low intracellular adenine concentration. The binding protein specific for UV-irradiated DNA (Feldberg, R.S., and Grossman, L. (1976) Biochemistry 15, 2402-2408) had no detectable purine or pyrimidine base insertion activity with depurinated or depyrimidinated DNAs.     

Purine and pyrimidine transport by cultured Novikoff cells. Specificities and mechanism of transport and relationship to phosphoribosylation.

Adenine, guanine, and hypoxanthine were rapidly incorporated into the acid-soluble nucleotide pool and nucleic acids by wild type Novikoff cells. Incorporation followed normal Michaelis-Menten kinetics, but the following evidence indicates that specific transport processes precede the phosphoribosyltransferase reactions and are the rate-limiting step in purine incorporation by whole cells. Cells of an azaguanine-resistant subline of Novikoff cells which lacked hypoxanthine-guanine phosphoribosyltransferase activity and failed to incorporate guanine or hypoxanthine into the nucleotide pool, exhibited uptake of guanine and hypoxanthine by a saturable process. Similarly, wild type cells which had been preincubated in a glucose-free basal medium containing KCN and iodoacetate transported guanine and hypoxanthine normally, although a conversion of these purines to nucleotides did not occur in these cells. The mutant and KCN-iodoacetate treated wild type cells also exhibited countertransport of guanine and hypoxanthine when preloaded with various purines, uracil, and pyrimidine nucleosides. The cells also possess a saturable transport system for uracil although they lack phosphoribosyltransferase activity for uracil. In the absence of phosphoribosylation, none of the substrates was accumulated against a concentration gradient. Thus transport is by facilitated diffusion (nonconcentrative transport). Furthermore, the apparent Km values for purine uptake by untreated wild type and azaguanine-resistant cells were higher and the apparent Vmax values were lower than those for the corresponding phosphoribosyltransferases...     

Study of purine degradation in aqueous solutions byParacoccus denitrificans

In this study the degradation of extracellular purines by the bacteriumParacoccus denitrificans was examined with aqueous purine solutions.Paracoccus denitrificans was able to decompose free purine bases and 5-mononucleotides. The nitrogen-containing products of the degradation were ammonia and urea. Purine uptake was the main control of purine decomposition. In the cases of guanine, xanthine, hypoxanthine, and urate, further control was exerted by induction. Furthermore, the uptake of the purines caused differences in the duration and temporal development of the substrate degration. It was also responsible for the inhibitory effects of the purines on the decomposition of one another when the substrates were used in mixtures. Also, fermentation parameters like biomass and purine concentration, pH, and temperature influenced the purine usage ofParacoccus denitrificans.     

Nitrite-induced deamination and hypochlorite-induced oxidation of DNA in intact human respiratory tract epithelial cells

No modification of purine or pyrimidine bases was observed when isolated DNA was incubated with 1 mM nitrite at pH 7.4. However, exposure of human bronchial epithelial cells in culture medium at pH 7.4 to nitrite at concentrations of 100 microM or greater led to deamination of purine bases in cellular DNA. Deamination was more extensive in cells exposed to lower extracellular pH values and higher nitrite concentrations. Significant increases in the levels of xanthine and hypoxanthine, putative deamination products of guanine and adenine, respectively, were observed in DNA from nitrite-treated cells but no rise in any base oxidation products such as 8-hydroxyguanine. This pattern of damage suggests that exposure of cells to nitrite (even at pH 7.4) leads to intracellular generation of "reactive nitrogen species" capable of deaminating purines in DNA. In addition, significant DNA strand breakage occurred in nitrite-treated cells. The time course of base damage suggested that the repair of deaminated purine lesions in these cells is slow. By contrast, DNA isolated from cells exposed to hypochlorous acid (HOCl) has significant oxidation of pyrimidine bases and chlorination of cytosine but little oxidation of purines. Exposure of cells to both species (NO(2)(-) plus HOCl) potentiated the oxidative DNA base damage observed but decreased the extent of deamination. We hypothesize that this is due to the formation of nitryl chloride (NO(2)Cl) from reaction of HOCl with *NO(2)(-). The relevance of our observations to events in the stomach and respiratory tract, at sites of inflammation, and in ischemic tissues is discussed.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced oxidative DNA damage (strand breaks and oxidized purines/pyrimidines) in human hepatoma cells

The aim of this study was to evaluate the effects of organosulfurs, isothiocyanates and vitamin C towards hydrogen peroxide-induced DNA damage (DNA strand breaks and oxidized purines/pyrimidines) in human hepatoma cells (HepG2), using the Comet assay. Treatment with hydrogen peroxide (H(2)O(2)) increased the levels of DNA strand breaks and oxidized purine and pyrimidine bases, in a concentration and time dependent manner. Organosulfur compounds (OSCs) reduced DNA strand breaks induced by H(2)O(2). In addition, OSCs also decreased the levels of oxidized pyrimidines. However, none of the OSCs tested reduced the levels of oxidized purines. Isothiocyanates compounds (ITCs) and vitamin C showed protective effects towards H(2)O(2)-induced DNA strand breaks and oxidized purine and pyrimidine bases. The results indicate that removal of oxidized purine and pyrimidine bases by ITCs was more efficient than by OSCs and vitamin C. Our findings suggest that OSCs, ITCs and vitamin C could exert their protective effects towards H(2)O(2)-induced DNA strand breaks and oxidative DNA damage by the free radical-scavenging efficiency of these compounds.     

Intracellular conversions of deoxyribonucleosides by Novikoff rat hepatoma cells and effects of hydroxyurea

The incorporation of ³H-labeled deoxyadenosine and deoxyguanosine into nucleic acids by cultured Novikoff rat hepatoma cells is about 80% into RNA and 20% into DNA. The pathways of incorporation have been elucidated in studies with whole cells and cell-free extracts. Deoxyadenosine is very rapidly deaminated to deoxyinosine. Most of the deoxyinosine formed by whole cells is transported out of the cells and accumulates in the medium. A portion of the deoxyinosine, and deoxyguanosine are phosphorolyzed by purine nucleoside phosphorylase to hypoxanthine and guanine, respectively. The latter are subsequently converted by hypoxanthine-guanine phosphoribosyl transferase to IMP and GMP, respectively. Incorporation of the purine deoxyribonucleosides into DNA is mainly via this pathway and the subsequent reduction of ADP and GDP by ribonucleoside reductase, although a small proportion of the deoxyadenosine and deoxyguanosine taken up by the cells seems to be directly phosphorylated to dAMP and dGMP, respectively. Deoxyguanosine is incorporated only into guanine residues of RNA and DNA. Deoxyadenosine is also mainly incorporated into guanine residues of RNA and DNA, although the radioactivity of deoxyadenosine in the acid-soluble pool is almost exclusively associated with ATP. A similar labeling pattern is observed with labeled deoxyinosine, inosine or hypoxanthine. The pyrimidine deoxyribonucleosides, on the other hand, are specific precursors for their respective bases in DNA. Hydroxyurea inhibits the incorporation of all deoxyribonucleosides into DNA. Results from pulse-chase experiments indicate that the inhibition of DNA synthesis is prevented by the presence of high concentrations of deoxyadenosine plus deoxyguanosine in the medium. Either purine deoxyribonucleoside alone or deoxycytidine, hypoxanthine or inosine alone or in combination with deoxyadenosine or deoxyguanosine are ineffective. The results are consistent with the conclusion that the inhibition of DNA synthesis is due to a depletion of the dATP and dGTP pools as a result of the hydroxyurea treatment. On the other hand, hydroxyurea causes an increased incorporation of thymidine and deoxycytidine into the dTTP and dCTP pools, respectively. Evidence is presented to indicate that this effect of hydroxyurea is due to an increased synthesis of dTTP and dCTP rather than to an inhibition of their turnover.     

Hematoporphyrin-induced photo-oxidation and photodynamic cross-linking of nucleic acids and their constituents

The pH-dependency of photo-oxidation of the physiological purine and pyrimidine bases and some of their derivatives was studied, with hematoporphyrin as sensitizer. At high pH these bases (adenine, guanine, uracil, thymine and cytosine) were photo-oxidizable. In the physiological pH range only guanine, and to a much less extent thymine, were sensitive to photo-oxidation. At physiological pH values a slow photo-oxidation of RNA and DNA took place. The photo-oxidation of nuclei acids was strongly augmented by perturbation of their structure in 8 M urea. In model experiments photodynamic cross-linking of tryptophan and cysteine to DNA was demonstrated. No covalent binding of purine or pyrimidine bases to DNA was observed. In similar model experiments covalent photodynamic coupling of guanosine and guanosine-monophosphate to proteins could be shown, whereas no coupling of the other bases occured. These studies confirm the preferential photo-oxidation of guanine in nucleic acids and demonstrate the possible photodynamic cross-linking of proteins to the guanine moiety in other molecules.