The guanine-hypoxanthine permease GhxP of Erwinia amylovora facilitates the influx of the toxic guanine derivative 6-thioguanine.

AIM: Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine transporter (EaGhxP) is required for virulence and if it can import the E. amylovora produced toxic analogue 6-thioguanine (6TG) into cells. METHODS AND RESULTS: Characterization of EaGhxP in guanine transport deficient Escherichia coli reveals that it can transport guanine, hypoxanthine and the toxic analogues 8-azaguanine (8AG) and 6TG. Similarly, EaGhxP transports 8AG and 6TG into E. amylovora cells. EaGhxP has a high affinity for 6TG with a Ki of 3·7 µmol l-1 . An E. amylovora ⊿ghxP::Camr strain shows resistance to growth on 8AG and 6TG. Although EaGhxP is expressed during active disease propagation, it is not necessary for virulence as determined on immature apple and pear assays. CONCLUSIONS: EaGhxP is not required for virulence, but it does import 6TG into E. amylovora cells. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of the disease establishment process, E. amylovora synthesizes and exports a toxic guanine derivative 6TG. Our results are counter intuitive and show that EaGhxP, an influx transporter, can move 6TG into cells raising questions regarding the role of 6TG in disease establishment.

Nitric oxide as a source for bacterial triazole biosynthesis.

The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.

New class of 8-aryl-7-deazaguanine cell permeable fluorescent probes.

A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.

Study of the interaction between 8-azaguanine and bovine serum albumin using optical spectroscopy and molecular modeling methods.

The interaction between 8-azaguanine (8-Azan) and bovine serum albumin (BSA) in Tris-HCl buffer solutions at pH 7.4 was investigated by means of fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. At 298 K and 310 K, at a wavelength of excitation (λ (ex)) of 282 nm, the fluorescence intensity decreased significantly with increasing concentrations of 8-Azan. Fluorescence static quenching was observed for BSA, which was attributed to the formation of a complex between 8-Azan and BSA during the binding reaction. This was illuminated further by the UV-Vis absorption spectra and the decomposition of the fluorescence spectra. The thermodynamic parameters ∆G, ∆H, ∆S were calculated. The results showed that the forces acting between 8-Azan and BSA were typical hydrophobic forces, and that the interaction process was spontaneous. The interaction distance r between 8-Azan and BSA, evaluated according to fluorescence resonance energy transfer theory, suggested that there is a high possibility of energy transfer from BSA to 8-Azan. Theoretical investigations based on homology modeling and molecular docking suggested that binding between 8-Azan and BSA is dominated by hydrophilic forces and hydrogen bonding. The theoretical investigations provided a good structural basis to explain the phenomenon of fluorescence quenching between 8-Azan and BSA.

Detection of pathogenic Leptospira from selected environment in Kelantan and Terengganu, Malaysia.

Leptospirosis is recognized as one of the important zoonotic diseases in the world including Malaysia. A total of 145 soil and water samples were collected from selected National Service Training Centres (NSTC) in Kelantan and Terengganu. The samples were inoculated into modified semisolid Ellinghausen McCullough Johnson Harris (EMJH) medium, incubated at room temperature for 1 month and examined under the dark-field microscope. Positive growth of the leptospiral isolates were then confirmed with 8-Azaguanine Test, Polymerase Chain Reaction (PCR) assay and Microscopic Agglutination Test (MAT). Fifteen cultures (10.34%) exhibited positive growths which were seen under dark field microscope whilst only 20% (3/15) were confirmed as pathogenic species. based on 8-Azaguanine Test and PCR. Serological identification of the isolates with MAT showed that hebdomadis was the dominant serovar in Terengganu. Pathogenic leptospires can be detected in Malaysian environment and this has the potential to cause an outbreak. Therefore, precautionary steps against leptospirosis should be taken by camp authorities to ensure the safety of trainees.

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis.

In plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine-guanine-hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine-guanine transporters.

Interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and a bisubstrate analogue inhibitor: implications for the reaction mechanism.

Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)-8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa approximately 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.

Interaction energy studies of an antimetabolite 8-azaguanine during transcription.

The possible incorporation of 8-azaguanine during transcription has been examined in the light of the model of transcription developed earlier by Sanyal et al. Electrostatic energy of interaction has been calculated for the nucleoside analogue (8-azaguanine) base for the entire space inside the deep groove of the DNA double helix. The interaction energy values and the location of the possible sites of association are compared with the recommended configurations of RNA transcription. It is concluded that 8-azaguanine is capable of replacing guanine during transcription. These conclusions are in general agreement with the experimental results.

Complementation analysis of locus for hypoxanthine guanine phosphoribosyltransferase in Chinese hamster cells.

The study deals with intragenic complementation between clones of Chinese hamster cells carrying mutations in the HPRT gene. All clones were of independent origin, selected in media containing one of three purine bases: 8-azaguanine (8 AG), 6-mercaptopurine (6MP), or 6-thioguanine (6TG). Some of the clones were spontaneous, others were induced by various mutagens. To make the study less time-consuming, an experimental set-up was proposed for simultaneous complementation testing of up to 10 clones. As a result, about 400 combinations of clones have been analyzed. Twelve pairs of complementating mutants have been identified in HAT medium. A linear complementation map has been constructed for the HPRT locus, showing five complementation groups. The changes in kinetic and other characteristics observed for mutant HPRT show that all the mutants studied carry structural gene mutations. Analysis of the biochemical characteristics of HPRT has revealed considerable differences between mutant enzymes in clones belonging to different complementation groups (three groups were examined). At the same time, the four mutant clones of complementation group II show similar HPRT characteristics, suggesting a relative similarity of their structural variants of the enzyme. The hybrid nature of HPRT in clones resulting from the fusion of mutant cells confirms the intragenic nature of complementation.

Isolation and characterization of human liver guanine deaminase.

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity.

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.

Guanine deaminase from rat brain. Purification, characteristics, and contribution to ammoniagenesis in the brain.

1. Guanine deaminase [EC 3.5.4.3] was purified to a homogeneous state from rat brain by a procedure involving ammonium sulfate fractionation, DE-52 column chromatography, hydroxylapatite column chromatography, gel filtration on ACA-34 and isoelectric focusing. Homogeneity was shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. 2. The molecular weight of the enzyme was determined by gel filtration (105,000), and that of its subunit by SDS-polyacrylamide gel electrophoresis (52,000). From these findings, we concluded that the native enzyme consisted of two identical subunits. 3. The Km values for guanine and 8-azaguanine were calculated to be 0.17 mM and 0.67 mM, respectively. This enzyme was markedly inhibited by 5-amino 4-imidazolecarboxamide (AICA), a precursor of purine nucleotide synthesis, with a K1 value of 82 micrometer. 4. Guanine deaminase was purified from rat liver by the procedure used for purification of the brain enzyme and anti-liver enzyme serum was raised in rabbits. This antiserum cross-reacted with the brain enzyme without spur formation in the Ouchterlony double diffusion test. 5. The gamma-globulin fraction of the anti-guanine deaminase serum and AICA inhibited more than 50% of the ammoniagenesis in the brain system in which the purine nucleotide cycle operates. It was also shown that guanine nucleotides were degraded via guanosine and guanine liberating ammonia in the same brain system when substrates for the purine nucleotide cycle were omitted. On the basis of these findings it is suggested that guanine deaminase contributes to ammoniagenesis in the brain.

Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in Novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation.

6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 +/- 23 microM; maximum velocity = 30 +/- 0.7 pmol/microl cell H2O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10-100 microM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pKa = 6.6) permeated the cell membrane. Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate.

Changes in amount of hypo-modified tRNA having guanine in place of queuine during erythroid differentiation of murine erythroleukemia cells.

The amounts of hypo-modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells. The amounts of these hypo-modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA--guanine transglycosylase. The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin. The amount of guanine-accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used. Decrease in the amounts of hypo-modified tRNAs in the cells was effectively blocked by 12-O-tetradecanoylphorbol 13-acetate, which inhibits differentiation of these cells. 8-Azaguanine, which is known to be substrate of tRNA--guanine transglycosylase, was incorporated almost exclusively into the first position of hypo-modified tRNA in murine erythroleukemic cells when they were pulse-labeled in culture with 8-azaguanine, suggesting strongly that tRNA-guanine transglycosylase in the cells is actually involved in incorporation of 8-azaguanine into tRNA in vivo. The amount of 8-azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine-accepting tRNA in these cells. The results suggest that the appearance of hypo-modified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. Par V. Radiation- and chemical carcinogen-induced mutagenesis.

8-Azaguanine(8AG)-resistant mutations induced by X-rays, ultraviolet radiation (UV) and a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) were examined during the cell cycle of synchronized HeLa S3 cells. Mutants induced by 400 R of X-rays occurred in a higher frequency in the X-ray-SENSITIVE Gl-S boundary phase than in the X-ray-resistant G1, S and early G2 phases. 8AG-resistant mutants induced by treatment with 10(-5) M 4-HAQO for 20 min appeared in a higher frequency in the early to middle S phases than in the other phases. In the case of UV, however, we found no significant difference in the induced mutation frequencies during the cell cycle, because the mutation frequencies induced by the UV doses (0-20 J/m2) used were too low for detection of the difference. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNA-synthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio-sensitivity.

Quantitative forward mutation assay in Salmonella typhimurium using 8-azaguanine resistance as a genetic marker.

We have developed a quantitative forward mutation assay using Salmonella typhimurium, in which resistance to the purine analog 8-azaguanine is used as a genetic marker. We present the assay protocol, the concentration-dependent toxicity and mutagenicity of five known mutagens (N-methyl-N'-nitro-N-nitrosoguanidine, ICR-191, 9-aminoacridine, dimethylnitrosamine, and benzo[a]pyrene), and reconstruction experiments testing the assay for possible bias. The relative merits of forward versus reverse mutation assays are discussed.

Lack of mutagenic effect of halothane or chloroform on cultured cells using the azaguanine test system.

Halothane with and without nitrous oxide and chloroform alone were tested for mutagenic effects at the 8-azaguanine locus on the chromosomes of Chinese hamster lung fibroblast cells in culture. No significant numbers of mutations were found after 24 h exposures to 1-3% of these anaesthetics, or to 75% nitrous oxide.