Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani

Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.     

Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl₂ for maximum activity but was inhibited by MnCl₂; its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K_m values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 μm and 11 μm; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5′-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5′-phosphate and 5-fluorouridine 5′-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.     

Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorpo?ation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells

It is presumed that the dGTP and dATP needed for replicative DNA synthesis can be formed by way of either `salvage' pathways or biosynthesis de novo. This was examined by adding hydroxyurea to cultures of rat thymus cells to inhibit ribonucleoside diphosphate reductase, a key enzyme of the `de novo' pathway. Most of the inhibition of the incorporation of [Me-³H]thymidine and deoxy[5-³H]cytidine by low concentrations of hydroxyurea (100–500μm) was prevented by substrates of the salvage pathway (400μm-deoxyguanosine and, to a lesser extent, 200μm-deoxyadenosine). However, isotope-dilution studies indicated that the purine deoxyribonucleosides prevented inhibition by decreasing pyrimidine deoxyribonucleotide competitor pools. Evidence was obtained that a hydroxyurea-induced increase in the thymidine-competitor pool (probably dTTP) was prevented to an equal extent by deoxyguanosine and by the inhibitor of thymidylate synthase, deoxy-5-fluorouridine. These compounds had almost identical effects on hydroxyurea dose–response curves and on thymidine isotope-dilution plots. The evidence suggests that exogenous purine deoxyribonucleosides cannot prevent the inhibition by hydroxyurea of thymus-cell DNA synthesis. This could mean that, with respect to the metabolism of purine deoxyribonucleotides, ribonucleoside diphosphate reductase is tightly coupled to DNA polymerase in a multienzyme complex. The complex would not permit entry of exogenous metabolic intermediates into the `de novo' pathway, but would still be subject to the regulatory effects of these intermediates. Thus dGTP and dATP formed from exogenous purine deoxyribonucleosides by salvage pathways might deplete pyrimidine deoxyribonucleotide competitor pools by inhibiting relatively hydroxyurea-insensitive activities of ribonucleoside diphosphate reductase.     

Pyrimidine Biosynthesis Is Not an Essential Function for Trypanosoma brucei Bloodstream Forms

Background

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.

Methodology/Principal Findings

Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5⁻/⁻ trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.

Conclusions/Significance

Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.     

The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH¹⁴CO₃ into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5–4-fold on incubation with 10mm-NH₄Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH¹⁴CO₃ into orotic acid was increased 3–4-fold in liver homogenate with NH₄Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH₄Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40–50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH₄Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH₄Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED₅₀ values in the 4-day murine P. berghei efficacy model of 13–21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.     

Genetical and biochemical evidence that a novel dinucleoside polyphosphate coordinates salvage and de novo nucleotide biosynthetic pathways in mammalian cells

Studies with ‘wild type’ Chinese hamster ovary cells and mutant derivatives defective in purine salvage and $\text{de novo}$ nucleotide biosynthesis pathways have brought to light the possibility that an unusual dinucleoside polyphosphate, HS-3 (see appendix) is a crucial regulator of these two pathways. Three antitumor drugs, methotrexate, 5-fluorouracil and azaserine as well as L-glutamine, purines and pyrimidines were used to define the loci of HS-3 metabolism. Wild type and salvage pathways mutants accumulated HS-3 in the absence of glutamine. $\text{De novo}$ pathways mutant accumulated HS-3 only when purine was absent. Depletion of HS-3 was induced in wild type and $\text{de novo}$ mutant cell lines by purine compounds. Salvage pathways mutants did not cause depletion of HS-3 when supplied with purines or pyrimidines, except 5-fluorouracil. Data indicate that HS-3 is probably synthesised when an early step in purine nucleotide synthesis is blocked and depleted when the salvage pathways are operative. HS-3 may be an important factor in certain diseases involving nucleotide metabolism.     

Evaluation of the antiprotozoan properties of 5′-norcarbocyclic pyrimidine nucleosides

Carbocyclic nucleoside analogues have a distinguished history as anti-infectious agents, including key antiviral agents. Toxicity was initially a concern but this was reduced by the introduction of 5′-nor variants. Here, we report the result of our preliminary screening of a series of 5′-norcarbocyclic uridine analogues against protozoan parasites, specifically the major pathogens Leishmania mexicana and Trypanosoma brucei. The series displayed antiparasite activity in the low to mid-micromolar range and establishes a preliminary structure-activity relationship, with the 4′,N³-di-(3,5-dimethylbenzoyl)-substituted analogues showing the most prominent activity. Utilizing an array of specially adapted cell lines, it was established that this series of analogues likely act through a common target. Moreover, the strong correlation between the trypanocidal and anti-leishmanial activities indicates that this mechanism is likely shared between the two species. EC₅₀ values were unaffected by the disabling of pyrimidine biosynthesis in T. brucei, showing that these uridine analogues do not act directly on the enzymes of pyrimidine nucleotide metabolism. The lack of cross-resistance with 5-fluorouracil, also establishes that the carbocyclic analogues are not imported through the known uracil transporters, thus offering forth new insights for this class of nucleosides. The lack of cross-resistance with current trypanocides makes this compound class interesting for further exploration.     

Tritrichomonas foetus: Partly defined cultivation medium for study of the purine and pyrimidine metabolism

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose—the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.     

A genetic and dietary study of the physiology of pyrimidine synthesis in Drosophila melanogaster

A combination of genetic and dietary manipulations have been utilized to investigate the physiology of pyrimidine metabolism throughout the Drosophila life cycle. We present evidence that the dietary sources of pyrimidines ingested at the larval stage are sufficient for all subsequent stages of the life cycle, except the process of oögenesis in adult females where a maternal supply of endogenously synthesized pyrimidines is normally required. Deprivation of dietary and de novo synthesis does not affect adult longevity; indicating that with the exception of oögenesis, all normal functions are carried out by recycling pyrimidines produced or ingested at the larval stage.In an enzymatic analysis, we have determined that Drosophila does not adjust for pyrimidine dietary deficiencies by significantly altering the level of synthesis of two tested enzymes encoded by the rudimentary locus, even though the pyrimidine deficiency is a rate limiting step in the completion of development.     

Uracil salvage is necessary for early Arabidopsis development

Uridine nucleotides can be formed by energy-consuming de novo synthesis or by the energy-saving recycling of nucleobases resulting from nucleotide catabolism. Uracil phosphoribosyltransferases (UPRTs; EC 2.4.2.9) are involved in the salvage of pyrimidines by catalyzing the formation of uridine monophosphate (UMP) from uracil and phosphoribosylpyrophosphate. To date, UPRTs are described as non-essential, energy-saving enzymes. In the present work, the six genes annotated as UPRTs in the Arabidopsis genome are examined through phylogenetic and functional complementation approaches and the available T-DNA insertion mutants are characterized. We show that a single nuclear gene encoding a protein targeted to plastids, UPP , is responsible for almost all UPRT activity in Arabidopsis. The inability to salvage uracil caused a light-dependent dramatic pale-green to albino phenotype, dwarfism and the inability to produce viable progeny in loss-of-function mutants. Plastid biogenesis and starch accumulation were affected in all analysed tissues, with the exception of stomata. Therefore we propose that uracil salvage is of major importance for plant development.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

Trypanosoma brucei: a survey of pyrimidine transport activities

Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as effective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identified a very high-affinity transporter for cytosine, designated C1, with a K(m) value of 0.048+/-0.009 microM. We also confirmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a K(m) of 33+/-5 microM. A higher-affinity U2 uridine transporter (K(m)=4.1+/-2.1 microM) was also identified, but efficiency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-fluorouracil was found to be effective. This drug was efficiently taken up by bloodstream forms of T. b. brucei.     

A Role for Purines and Pyrimidines of Ribonucleic Acid in the Induction of Two-dimensional Growth in the Gametophytes of the Fern, Asplenium nidus

The inhibition of two-dimensional growth in the gametophytesof Asplenium nidus induced by purine and pyrimidine analoguesand the reversal of inhibition by natural purine and pyrimidinebases and their derivatives have been studied. Adenine and guanineand their ribosides and ribotides were more effective than cytosine,uracil, thymine, and their derivatives in preventing the inhibitiondue to 8-azaadenine and 8-azaguanine. Likewise, the inhibitoryeffects of 2-thiocytosine, 2-thiouracil,6-azauracil, and 5-fluorouracilwere overcome by the pyrimidines and their derivatives, butnot usually by the purines.Combinations of two purine analoguesor two pyrimidine analogues or one purine analogue and one pyrimidineanalogue inhibited growth more effectively than single compounds.The combined inhibitions were maximally reversed when both naturalbases or their derivatives were added to the medium. It is concludedthat there is a requirement for both purines and pyrimidinesof ribonucleic acid in the induction of two-dimensional growthin the gametophytes of Asplenium nidus.     

5-Oxoprolinase (l-Pyroglutamate Hydrolase) in Higher Plants: Partial Purification and Characterization of the Wheat Germ Enzyme

5-Oxoprolinase has been found to be widely distributed in higher plants. This enzyme catalyzes the ATP-dependent hydrolysis of 5-oxo-l-proline (l-pyrollidone carboxylate, l-pyroglutamate) to glutamate. The enzyme has been purified almost 60 fold from wheat germ (Triticum aestivum L). This enzyme requires a divalent cation, either Mn²⁺ or Mg²⁺, and a combination of both appears to be the most effective. There is also an absolute requirement for a monovalent cation best fulfilled by either NH₄⁺ or K⁺. The K_m for ATP is 0.4 mm and for 5-oxo-l-proline is 14 μm. A small amount of activity is observed when other purine nucleotides such as ITP and GTP replace ATP. The substitution of the pyrimidine nucleotides CTP and UTP for ATP yield almost completely inactive preparations. The enzyme appears to have an active sulfhydryl group since there is an increase in activity in the presence of dithioerythritol. Preincubation with reagents such as N-ethylmaleimide or iodoacetamide lead to complete inactivation. The presence of this enzyme leads to the speculation of the possible presence of a γ-glutamyl cycle in higher plants.     

Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

The Crystal Structure and Activity of a Putative Trypanosomal Nucleoside Phosphorylase Reveal It to be a Homodimeric Uridine Phosphorylase

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.