A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani

Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non‐permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine‐containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long‐term survival of Leishmania in a purine‐scarce environment.     

AUXIN-CYTOKININ CONTROL OF SECONDARY VASCULAR TISSUE FORMATION IN ISOLATED ROOTS OF RAPHANUS

A comparative study was made of the effectiveness of various hormone and metabolite mixtures in inducing vascular cambium initiation and secondary vascular tissue formation in isolated first-transfer roots of the radish, Raphanus sativus L. ‘White Icicle,‘ when provided to the cut basal end of the root grown in sterile culture. An auxin, such as indoleacetic acid (IAA) at 10^–5 m , a cytokinin, such as 6-benzylamino purine at 5 × 10^–6 m , a cyclitol, such as myo-inositol at 5 × 10^–4 M and sucrose at 8% were all required for maximum response. Requirements for auxin and cytokinin were absolute; in their absence no cambium was formed. The addition of cyclitol, while not an absolute requirement for cambium initiation, increased the magnitude of the response markedly. Alternative auxins such as α-naphthaleneaeetic acid and 2,4-dichlorophenoxyacetic acid were equally as effective as IAA. Alternative effective cytokinins included 6-furfurylaminopurine, 6-phenylaminopurine and 6-(γ,γ-dimethylallylamino)purine. Alternative cyclitols equivalent to myo-inositol were seyllitol and pinitol. Other related cyclitols tested were much less effective or totally inactive.     

SYNTHESIS AND EVALUATION OF ANTIVIRAL ACTIVITY OF HIGHER HOMOLOGUES OF XYLO-CARBOCYCLIC NUCLEOSIDES

New carbocyclic nucleosides with purine (compounds 3a and 3b), and 8-azapurine (compounds 3c and 3d) as base were prepared and assayed for in vitro activity.     

Purine metabolism and the microaerophily of Helicobacter pylori

The requirements for purine nucleotide synthesis, the effects of purine analogues, and the metabolism of adenine in the bacterium Helicobacter pylori were investigated employing cell culture techniques and one-dimensional NMR spectroscopy. Bacterial cells grew and proliferated in media lacking preformed purines, indicating that H. pylori can synthesize purine nucleotides de novo to meet its requirements. Blocking of this pathway in the absence of sufficient preformed purines for salvage nucleotide synthesis led to cell death. Analogues of purine nucleobases and nucleosides taken up by the cells were cytotoxic, suggesting that salvage routes could be exploited for therapy. Adenine or hypoxanthine were able to substitute for catalase in supporting cell growth and proliferation, suggesting a role for these bases in maintaining the microaerophilic conditions essentially required by the bacterium. Received: 23 May 1997 / Accepted: 17 July 1997     

Microbial Synthesis of Purine Arabinosides and Their Biological Activity

A simple method for the synthesis of various purine arabinosides from purine bases and uracil arabinoside by microbial transarabinosylation is described. A wet cell paste of Enterobacter aerogenes AJ 11125 showed a wide substrate specificity range for purine bases. Not only naturally occurring purine bases such as adenine and hypoxanthine but also unnatural bases such as 6-thioguanine and 2-chlorohypoxanthine were catalyzed to give the corresponding purine arabinosides. The enzymatically synthesized purine arabinosides were isolated from the reaction mixtures and identified by physicochemical means. The biological activities of the compounds were investigated and it was found that thioguanine arabinoside and 2-methyladenine arabinoside have potent activity against Hela cells, and their ED₅₀ were 10.5 and 21.5 μg/ml, respectively.     

Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

5′-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous purine compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented at during growth of B. cereus in the presence of AMP, the concerted action of 5′-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B cereus acts as a translocase of the ribose moiety of ionsine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905–7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Oxidative degradation of purines by the faculative phototrophic bacterium Rhodopseudomonas capsulata

The mechanism of purine degradation was studied in the facultative phototrophic bacterium Rhodopseudomonas capsulata. Using tungstate as an inhibitor of synthesis of an active xanthine dehydrogenase it could be shown in growth experiments that purine compounds are transformed to uric acid as central purine intermediate prior to ring cleavage. Because of its rapid degradation, the mechanism of uric acid conversion was investigated using 1-methyluric acid as substrate. The analogue was partially degraded by whole cells yielding 3-methylallantoin and methylurea. This implicated an oxidative degradation of 1-methyluric acid analogous to oxidation of uric acid to allantoin suggesting uric acid degradation via allantoin. In cell-free extracts, allantoinase, allantoicase, ureidoglycolase and urease activities degrading allantoin to NH₃, CO₂ and glyoxylic acid were detected. Apparently, purine degradation in R. capsulata proceeds in a manner similar to many aerobic microorganisms. It is peculiar to this bacterium, however, that the pathway evidently operates also under anaerobic conditions. In cell extracts, oxidation of uric acid was observed which could be increased by addition of cytochrome c. The basis of this stimulation is still unknown.     

Differentiation of adenine non-planarity in valence molecular orbitals

Two molecular orbitals: MO7 (29a) and MO13 (23a) have been identified using dual space analysis (DSA) as the signatures of adenine non-planarity (C₁ point group symmetry). The non-planarity of adenine has been demonstrated to be from the attachment of the amino group (NH₂) to purine rings as well as the non-rigid deformability of the purine ring of adenine. Orbital 29a (3a″ in the planar case), a π-like orbital, is the direct result of the attachment of the amino group to the purine ring. Orbital 23a (23a′ in the planar case) is the result of the deformability of the purine ring in non-planar adenine (NP) and will be experimentally challenging to resolve.     

METABOLIC EFFECTS OF AZASERINE IN RAT BRAIN

The de novo biosynthesis of purine nucleotides in rat brain was markedly and rapidly inhibited by intracerebral injections of azaserine. After 48 hr the animals became paraplegic. The biosynthesis of purine and pyrimidine nucleotides, RNA and protein was investigated by radioactive tracer studies over a 4-day period in an attempt to correlate the azaserine-induced, metabolic lesions found in rat brain and the manifested, peripheral, neurological disturbance.     

Reversed-phase high-performance liquid chromatography of purine compounds for investigation of biomedical problems: application to different tissues and body fluids

An overview of high-performance liquid chromatographic separation techniques (reversed-phase and ion-pair reversed-phase) used in the analysis of purine ribonucleotides, ribonucleosides and nucleobases, including procedures for sample preparation, is given. Coverage of the separation techniques is extended to the measurement of specific radioactivities of these compounds in tracer kinetic experiments for metabolic flux rate analyses. This article is focused on the development and adaptation of reversed-phase separation techniques for nucleotides, nucleosides and bases that are used to examine a variety of biomedical problems. The investigation of purine nucleotide metabolic disorders or physiological transition in the pathomechanisms of different diseases and syndromes or in cell maturation processes, respectively, requires the application of chromatographic separation to a multitude of tissues and body fluids. These samples vary greatly in concentrations of purine compounds with low molecular mass, from ca. 5 mM to ca. 0.5 μM. The advantages and disadvantages of different techniques are critically discussed.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates

Using we11-characterized mutant host cell lines, deficient in specific enzymes of energy and nucleotide metabolism, we addressed numerous questions regarding nucleotide metabolism in the obligate intracellular bacterium Chlamydia trachomatis. The results presented indicate that C. trachomatis: (i) does not absolutely depend on mitochondrial generated ATP for survival; (ii) does have a significant draw on host-cell NTP pools but does not have a detrimental effect on the ability of the host cell to maintain its energy charge; (iii) lacks the ability to synthesize purine and pyrimidine nucleotides de novo; (iv) is not capable of interconverting purine nucleotides; and (v) possesses the pyrimidine metabolic-pathway enzymes CTP synthetase and deoxycytidine nucleotide deaminase. In total our results indicate that C. trachomatis is auxotrophic for host-cell ATP, GTP and UTP. In contrast, CTP can be obtained from the host cell or it can be synthesized from UTP by the parasite.     

Purine Regulon of Gamma-Proteobacteria: A Detailed Description

The structure of the purine regulon was studied by a comparative genomic approach in seven genomes of gamma-proteobacteria: Escherichia coli, Salmonella typhimurium, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Vibrio cholerae. The palindromic binding site of the purine repressor (consensus ACGCAAACGTTTGCGT) is fairly well conserved upstream genes encoding enzymes that participate in the synthesis of inosine monophosphate from phosphoribozylpyrophosphate and in transfer of one-carbon units, and also upstream of some transport protein genes. These genes may be regarded as the main part of the purine regulon. In terms of physiology, the regulation of the purC and gcvTHP/folD genes seems to be especially important, because the PurR site was found upstream nonorthologous but functionally replaceable genes. However, the PurR site is poorly conserved upstream orthologs of some genes belonging to the E. coli purine regulon, such as genes involved in general nitrogen metabolism, biosynthesis of pyrimidines, and synthesis of AMP and GMP from IMP, and also upstream of the purine repressor gene. It is predicted that purine regulons of the examined bacteria include the following genes: upp participating in synthesis of pyrimidines; uraA encoding an uracil transporter gene; serA involved in serine biosynthesis; folD responsible for the conversion of N5,N10-methenyl tetrahydrofolate into N10-formyltetrahydrofolate; rpiA involved in ribose metabolism; and genes with an unknown function (yhhQ and ydiK). The PurR site was shown to have different structure in different genomes. Thus, the tendency for a decline of the conservatism of site positions 2 and 15 was observed in genomes of bacteria belonging to the Pasteurellaceae and Vibrionaceae groups.     

Cloning,expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8

Abstract

Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5′-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg²⁺. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90?°C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis.     

PHOSPHONATE ANALOGS OF CARBOCYCLIC PHOSPHORIBOSYLAMINE AND CARBOCYCLIC GLYCINAMIDE RIBONUCLEOTIDE

Abstract

Analogs of intermediates in the de novo purine nucleotide biosynthetic pathway were synthesized to study the binding requirements of the corresponding enzymes. Because of the instability of the natural stubstrates, such as phosphoribosylamine, the use of the structurally stable phosphonate moiety and the carbocyclic ribose yields ideal analogs for these studies. In addition, these analogs can act as potential inhibitors of the de novo pathway leading to the design of anticancer agents. Enzyme studies with GAR synthetase and GAR transformylase reveal that the title compounds can act as substrates or inhibitors of the de novo enzymes.     

RESOLUTION OF RACEMIC MIXTURES OF CARBOCYCLIC ANALOGUES OF NUCLEOSIDES AND ASSIGNMENT OF THEIR ABSOLUTE CONFIGURATION

Racemic trans-6-chloro-9-[2-(hydroxymethyl)cyclopentyl]purine was resolved using HPLC with a chiral column. The absolute configurations of the enantiomers were determined by NMR studies of their (R)- and (S)-methoxy-phenylacetates.     

EFFECTS OF PURINES,PYRIMIDINES AND RELATED COMPOUNDS UPON CELL FORM IN USTILAGO SPHAEROGENA

Spoerl , Edward , and J. L. Pullman . (U. S. Army Med. Res. Lab., Fort Knox, Ky.) Effects of purines, pyrimidines and related compounds upon cell form in Ustilago sphaerogena. Amer. Jour. Bot. 46(9): 651–656. 1959.—Nucleotide, nucleoside and base constituents of ribosenucleic acid, singly and in combination, were used as nitrogen sources for U. sphaerogena and examined for their effects upon cell form. Combinations of nucleotides, and adenine, produced long cells, i.e., interfered with cell division. A variety of analogues, especially of purine types, was examined for their effects upon cell form. Although adenine produced long cells, a hydroxy group substituted at the 6 position of the purine ring produced short cells. Sulfhydryl, furfurylamino and methyl groups substituted at the 6 position produced long cells. Additional groups and combinations of these groups also were examined. Neither hydroxypurines nor methionine or norleucine, which normally promote short-cell formation, effectively antagonized the adenine effect, although norleucine tended to counteract a dimercaptopurine effect.     

Microbial Synthesis of Purine 2′-Amino-2′-deoxyribosides

The microbial synthesis of some purine 2′-amino-2′-deoxyribonucleosides from purine bases and 2′-amino-2′-deoxyuridine is described. Various bacteria, especially Erwinia herbicola, Salmonella schottmuelleri, Enterobacter aerogenes and Escherichia coli, were able to transfer the aminoribosyl moiety of 2′-amino-2′-deoxyuridine to purine bases (transaminoribosylation) in the presence of inorganic phosphate. The optimum conditions for the reaction were pH 7.0 and 63°C. No reaction was observed in the absence of inorganic phosphate and the optimum concentration of it was around 30 mm. Adenine, guanine, 2-chlorohypoxanthine and hypoxanthine were transformed to the corresponding 2′-amino-2′-deoxyribonucleosides by the catalytic activity of the wet cell paste of Enterobacter aerogenes AJ 11125. The enzymatically synthesized purine 2′-amino-2′-deoxyribonucleosides were isolated and identified by physicochemical means. 2′-Amino-2′-deoxyadenosine strongly inhibited the growth of Hela cells in tissue culture, and the ED⁵⁰ was 2.5μ/ml.