Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani

Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP(i). Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis for catalysis (Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J. 18, 3533-3545). To address this issue, a steady state and transient kinetic analysis of the enzyme was performed in order to determine the catalytic mechanism. Initial velocity and product inhibition studies indicated that LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind and AMP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchange and fluorescence binding studies, which provided dissociation constants for the LdAPRT-PRPP and LdAPRT-AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed a burst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement of rates of binary complex dissociation that implicated AMP release as rate-limiting for the forward reaction. Kinetics of product ternary complex formation were evaluated using the fluorophore formycin AMP and established rate constants for pyrophosphate binding to the LdAPRT-formycin AMP complex. Taken together, these data enabled the complete formulation of an ordered bi-bi kinetic mechanism for LdAPRT in which all of the rate constants were either measured or calculated.     

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite.

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine–inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca²⁺-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-β- -ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.     

A novel role for 100 kD heat shock proteins in the parasite Leishmania donovani

Heat shock proteins of the 100 kD family have been known to confer general stress tolerance in yeast and plants. Several protozoan parasites possess genes for Hsp100 proteins. In Leishmania species the protein is expressed under heat stress and during the mammalian stage, the amastigote. We show here that replacement of the clpB gene which encodes Hsp100 does not affect thermotolerance or general viability in Leishmania donovani insect stages (promastigotes) nor in axenically cultured mammalian stages (amastigotes). However, its expression is required for normal development of the parasite inside mammalian host cells. Hsp100 appears to function as an antagonist of amastigote-to-promastigote differentiation and a promoter of full amastigote development.     

Adenine aminohydrolase from Leishmania donovani: unique enzyme in parasite purine metabolism

Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K(m) of 15.4 μM, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani.     

A conditional mutant deficient in hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase validates the purine salvage pathway of Leishmania donovani

Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional delta hgprt/delta xprt mutant strain that exhibits an absolute requirement for 2'-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the delta hgprt/delta xprt strain cannot proliferate indefinitely without 2'-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The delta hgprt/delta xprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates.     

Phosphofructokinase of Leishmania donovani and Leishmania braziliensis and its role in glycolysis.

It was shown in an investigation of the phosphofructokinases of Leishmania donovani and Leishmania braziliensis that both enzymes are similar to that of Crithidia fasciculata. Although the enzymes are allosteric with respect to their substrates and require AMP for activation, there is no influence by other heterotropic modifiers. The Mg2+-ATP chelate activates these enzymes in a first order process and they can be inhibited by free ATP. The inhibition is reversed by the activator, AMP, in a competitive manner. The requirement for the nucleotide in L. donovani can be eliminated by decreasing the pH. The data indicate that phosphofructokinase, a pivotal enzyme in glycolysis for most organisms, probably does not play an important role in glycolysis in Leishmania.     

A role for oxygen-dependent mechanisms in killing of Leishmania donovani tissue forms by activated macrophages

Leishmania donovani, the causative agent of visceral leishmaniasis, infects macrophages (M phi ) of susceptible vertebrates. Immunologically activated M phi are leishmanicidal, but the mechanisms involved in the killing process are not well defined. We sought to investigate the role of reactive oxygen intermediates in the killing of L. donovani. Both the free-swimming promastigote and the intracellular amastigote forms were found to be susceptible to killing in vitro by hydrogen peroxide and other oxygen intermediates. Upon phagocytosis by mouse peritoneal M phi, promastigotes elicited a significantly stronger respiratory burst compared with amastigotes as measured by release of superoxide anion. Although amastigotes do not elicit a strong burst of M phi oxidative metabolism during the initial phagocytic event, immunologically activated M phi that acquired leishmanicidal capacity could be triggered to release substantial amounts of H2O2. Hence, the development of leishmanicidal capacity was correlated temporally with enhanced H2O2 generation by the M phi. In contrast, M phi that lost their ability to release significant amounts of H2O2 after several days in culture were unable to eliminate their parasite burden. Catalase markedly inhibited the elimination of amastigotes by lymphokine-stimulated M phi. In toto, the results implicate reactive oxygen intermediates in killing of the tissue form of L. donovani by its host cell, the mononuclear phagocyte.     

Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

Purinergic Signalling - The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism...     

Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase

Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.     

A cyclic nucleotide-independent protein kinase in Leishmania donovani.

Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase.     

A novel active DNA topoisomerase I in Leishmania donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.     

Systematic variations in the content of the purine nucleotides in the steady-state perfused rat heart. Evidence for the existence of controlled storage and release of adenine nucleotides

1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1μg/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3μmol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem. 250, 6304–6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.     

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5'- and 3'-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8-(14)C]adenosine and [8-(14)C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.     

Possibility of membrane modification as a mechanism of antimony resistance in Leishmania donovani

Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resistance whose localization in the present study was confirmed on the pellicular plasma membrane of the parasite thereby indicating towards membrane modification as a mechanism of resistance in this resistant isolate.