Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasite's host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.     

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.     

Purification of hypoxanthine-guanine phosphoribosyltransferase of Plasmodium lophurae

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was isolated from the malarial parasite, Plasmodium lophurae. The apparent pI, as determined by chromatofocusing, was 7.6. The native molecular weight was 79,000. The pH profile of HGPRT exhibited a broad pH optimum. With hypoxanthine as substrate maximal activity was achieved from pH 6.0-10.0, and with guanine as substrate maximal activity occurred from pH 7.5-9.5. The enzyme exhibited Michaelis-Menten kinetics with all substrates. The Km values were 3.8 microM (hypoxanthine), 2.4 microM (guanine), 6.2 microM (6-mercaptopurine), 7.6 microM (6-thioguanine), and 360 microM (8-azahypoxanthine). 6-Thioinosine, 9-beta-arabinofuranosylhypoxanthine, 6-chloropurine, xanthine and azaguanine were inhibitors of the P. lophurae enzyme. From the substrate and inhibitor data it appears that the sixth position on the purine ring plays a major role in enzyme activity.     

Lesch-Nyhan syndrome: mRNA expression of HPRT in patients with enzyme proven deficiency of HPRT and normal HPRT coding region of the DNA

Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome (LNS) or Lesch-Nyhan variants (LNV). We report a case of two LNS affected members of a family with deficiency of activity of HPRT in intact cultured fibroblasts in whom mutation could not be found in the HPRT coding sequence but there was markedly decreased HPRT expression of mRNA.     

The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties

Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyl-transferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyl-transferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [¹⁴C]hypoxanthine, [¹⁴C]guanine or [¹⁴C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 μM, 3.8 μM and 52.4 μM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodiumfalciparum, which are dimers, with xanthine about 100-times less proficient as a substrate. T. foetus HGXPRTase is thus a unique enzyme with opportunity for specific inhibitor design.     

Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

Purine metabolism in Trypanosoma cruzi

Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rate-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine > hypoxanthine > guanine > xanthine.     

Abnormalities in cellular adhesion of neuroblastoma and fibroblast models of Lesch Nyhan syndrome

Lesch Nyhan syndrome is a neurological paediatric condition characterized by mental retardation, choreathotosis and self-mutilation. Biochemically, this condition has been attributed to a deficiency in the purine enzyme, hypoxanthine guanine phosphoribosyltransferase, however, the way this affects the development of the nervous system is still unknown. Ma et al.(15) and Stacey et al.(25) found that hypoxanthine guanine phosphoribosyltransferase-deficient neuroblastoma, differentiated significantly more than cells with this enzyme. Here, we report that adhesion of hypoxanthine guanine phosphoribosyltransferase-deficient neuroblastoma as well as fibroblasts from patients with Lesch Nyhan syndrome, exhibited dramatically enhanced adhesion compared to control cells. This increase in adhesion was dependent upon the cell type, density of the cells and upon the substrate used.Development of the nervous system is dependent on adhesion, in particular in the processes of migration, nucleation, differentiation and fasciculation. Our results suggest that the increased adhesion of hypoxanthine guanine phosphoribosyltransferase-deficient neuroblastoma and fibroblasts in vitro underpins the neuropathology of Lesch Nyhan syndrome.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.     

Leishmania donovani singly deficient in HGPRT, APRT or XPRT are viable in vitro and within mammalian macrophages

Leishmania species express three phosphoribosyltransferase enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and xanthine phosphoribosyltransferase (XPRT), which enable this genus to acquire purine nutrients from their hosts. To test whether any of these enzymes is essential for viability, transformation into amastigotes, and infectivity and proliferation within mammalian macrophages, Deltahgprt, Deltaaprt, and Deltaxprt null mutants were created by targeted gene replacement within a virulent background of Leishmania donovani. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in bone marrow-derived murine macrophages. These data support the hypothesis that none of the three phosphoribosyltransferases is essential for purine salvage or viability by itself and that purine salvage occurs through multiple anabolic routes in both parasite life cycle stages. In addition these studies revealed the presence of an adenine aminohydrolase enzyme in L. donovani axenic amastigotes, an activity previously thought to be restricted to promastigotes.     

Purine and pyrimidine metabolizing activities in Trichomonas vaginalis extracts

Sixty-one purine and pyrimidine metabolizing activities were assayed in extracts of Trichomonas vaginalis. Of these, 43 were detected and quantitated. The only phosphoribosyltransfer activity observed was with uracil. No such activity was observed with adenine, guanine, hypoxanthine, xanthine or orotic acid. The rate of nucleoside cleavage was increased dramatically by the addition of inorganic phosphate. In addition, the extracts could catalyze the synthesis of ribonucleosides from the bases adenine, hypoxanthine, guanine and uracil but not cytosine, thymine or orotic acid, in the presence of ribose 1-phosphate. These data suggest that T. vaginalis contains primarily nucleoside phosphorylases instead of nucleoside hydrolases. Adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, uridine, thymidine and GMP were phosphorylated in the presence of ATP. No nucleoside phosphotransferase activity was detected. Deamination of guanine, adenosine, deoxyadenosine, cytidine and deoxycytidine but not adenine was observed. These data suggest that salvage of adenine and guanine for ribonucleotide synthesis in T. vaginalis occurs via a phosphorylase/kinase pathway instead of through a phosphoribosyltransferase pathway which predominates in mammalian cells. In contrast, the pyrimidine base uracil can be converted to UMP via both a phosphoribosyltransferase or a phosphorylase/kinase pathway, analogous to that in mammals.     

Purine salvage and metabolism inBabesia bovis

Studies of the incorporation of radio-labelled purine precursors into the erythrocytic forms ofBabesia bovis under tissue-culture conditions have confirmed the presence in the parasite of enzymatic activities responsible for the salvage of preformed purines. The results also revealed that the parasite was capable of a variety of nucleotide interconversions, such that exogenous hypoxanthine and adenosine were incorporated into both adenine and guanine nucleotides followed by the incorporation of these nucleotides into the adenine and guanine moieties of RNA and DNA. No evidence was found for salvage of preformed pyrimidines. Evidence was also obtained for the insertion of a parasite-specific nucleoside/nucleobase transporter into the membrane of the bovine (host) red cell. Thus, whereas normal (non-parasitised) bovine red cells are essentially incapable of transporting nucleosides across their membranes, the invasion of these cells byB. bovis introduces a transporter that can be inhibited by classic nucleoside transport inhibitors.     

Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

Exogenous guanine was found to be incorporated into the nucleic acids of Chlamydia psittaci when the parasite was grown in HeLa cells containing hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) activity but not when the parasite was grown in transferase-deficient HeLa cells. No evidence for a chlamydia-specific transferase activity was found in either transferase-containing or transferase-deficient infected HeLa cells. It is concluded that C. psittaci is incapable of metabolizing guanine, but that the parasite can use host-generated guanine nucleotides as precursors for nucleic acid synthesis.     

Adenosine is the primary precursor of all purine nucleotides in Trichomonas vaginalis

Trichomonas vaginalis, a parasitic protozoan and the causative agent of trichomoniasis, lacks de novo purine nucleotide synthesis and possesses a unique purine salvage pathway, consisting of a bacterial type purine nucleoside phosphorylase and a purine nucleoside kinase. It is generally believed that adenine and guanine are converted to their corresponding nucleosides and then further phosphorylated to form AMP and GMP, respectively, as the main as well as the essential pathway of replenishing the purine nucleotide pool in the organism. Formycin A, an analogue of adenosine, inhibits both enzymes as well as the in vitro growth of T. vaginalis with an estimated IC(50) of 0.27 microM. This growth inhibition was reversed by adding adenine to the culture medium but not by adding guanine or hypoxanthine. Furthermore, T. vaginalis can grow in semi-defined medium supplemented with only adenine but not with guanine or hypoxanthine. Radiolabeling experiments followed by HPLC analysis of the purine nucleotide pool in T. vaginalis demonstrated incorporation of [8-14C]adenine into both adenine and guanine nucleotides, whereas [8-14C]guanine was incorporated only into guanine nucleotides. Substantial adenosine deaminase activity and significant IMP dehydrogenase and GMP synthetase activities were identified in T. vaginalis lysate, suggesting a pathway capable of converting adenine to GMP via adenosine. This purine salvage scheme depicts adenosine the primary precursor of the entire purine nucleotide pool in T. vaginalis and the purine nucleoside kinase one of the most pivotal enzymes in purine salvage and a potential target for anti-trichomoniasis chemotherapy.     

The gene for hypoxanthine phosphoribosyl transferase of Plasmodium falciparum complements a bacterial HPT mutation

The enzyme hypoxanthine phosphoribosyl transferase of Plasmodium falciparum has been overexpressed in Escherichia coli. The protein was found to be active enzymatically. When the recombinant expression vector (pPfPRT2) was transformed and expressed in a Salmonella typhimurium mutant KP1684 (purE deoD hpt gpt), the active expressed protein complemented the hpt mutation in the bacteria. We discuss the practical value of this strain. Assays of the expressed protein in the mutant extract showed that the enzyme is able to use hypoxanthine, guanine and xanthine as substrates. A specificity study using the competitive inhibitor, 6-thioguanine, showed that of these hypoxanthine is the most favourable substrate. The biological significance of xanthine utilisation by the enzyme is discussed.     

Mechanism of growth inhibition of intraerythrocytic stages of Plasmodium falciparum by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)

Purine nucleotide synthesis in Plasmodium falciparum takes place solely by the purine salvage pathway in which preformed purine base(s) are salvaged from the host and acted upon by a battery of enzymes to generate AMP and GMP. Inhibitors of this pathway have a potent effect on the in vitro growth of P. falciparum and are hence, implicated as promising leads for the development of new generation anti-malarials. Here, we describe the mechanism of inhibition of the intraerythrocytic growth of P. falciparum by the purine nucleoside precursor, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Our results show that AICAR toxicity is mediated through the erythrocyte in which AICAR is phosphorylated to its nucleotide, ZMP. Further, purine metabolite labeling of the parasitized erythrocytes by [(3)H]-hypoxanthine, in the presence of AICAR, showed a significant decrease in radioactive counts in adenylate fractions but not in guanylate fractions. The most dramatic effect on parasite growth was observed when erythrocytes pretreated with AICAR were used in culture. Pretreatment of erythrocytes with AICAR led to significant intracellular accumulation of ZMP and these erythrocytes were incapable of supporting parasite growth. These results implicate that in addition to the purine salvage pathway in P. falciparum, AICAR alters the metabolic status of the erythrocytes, which inhibits parasite growth. As AICAR and ZMP are metabolites in the human serum and erythrocytes, our studies reported here throw light on their possible role in disease susceptibility, and also suggests the possibility of AICAR being a potential prophylactic or chemotherapeutic anti-malarial compound.     

Purine base transport in wild-type and mycophenolic acid-resistant Tritrichomonas foetus

The purine base transport systems of wild-type and mycophenolic acid-resistant (MPAR) Tritrichomonas foetus have been characterized. Wild-type T. foetus has two carriers, one for hypoxanthine (Km = 0.7 +/- 0.3 mM, Vm = 80 +/- 20 pmol microliters-1min-1) and guanine (Km = 0.09 +/- 0.02 mM, Vm = 17 +/- 3 pmol microliters-1min-1), and a second for xanthine (Km = 0.6 +/- 0.2 mM, Vm = 25 +/- 5 pmol microliters-1min-1). Adenine transport was not saturable (k = 0.16 +/- 0.01 min-1) and therefore appears to enter the parasite by passive diffusion through the membrane. T. foetus MPAR has lost the hypoxanthine/guanine transporter. Xanthine and adenine transport are similar in wild-type and MPAR T. foetus. No purine nucleoside transporter could be identified.     

Purine metabolism by the avian malarial parasite Plasmodium lophurae

Extracts of normal duckling erythrocytes catabolized AMP to IMP, inosine and hypoxanthine; adenosine and adenine were not formed from AMP. When erythrocyte-free Plasmodium lophurae, prepared by antibody lysis, were incubated in the presence of [¹⁴C]hypoxanthine approximately 60% of the label was recovered as purine nucleotides and there was no evidence for extracellular alteration of added hypoxanthine. However, when adenosine was added to suspensions of antibody- or saponin-prepared parasites extensive conversion to inosine and hypoxanthine occurred. This conversion was found to be the result of parasite lysis with release of cytosolic purine salvage pathway enzymes; plasmodial surface membrane ecto-enzymes were not responsible for adenosine catabolism. It appears that in vivo the intracellular plasmodium utilizes the normal erythrocytic process of purine turnover to avail itself of hypoxanthine, the red cell's end-product, and at the same time the parasite avoids direct competition for adenosine essential to erythrocyte survival. Since the blood plasma of infected ducklings contained increased amounts of hypoxanthine it is possible that P. lophurae also utilizes this as a purine source.