Abstract
The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent Km values of 20 and 99 μM for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent Km values 6 and 7 μM for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.
Highlights
Leishmania donovani salvage all pyrimidines through uracil phosphoribosyltransferase (LdUPRT)
Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and phosphoribosylated to UMP in the parasite by LdUPRT
The purified LdUPRT was stable with no measurable loss of enzymatic activity for Ͼ1 month when maintained at 4 °C in the elution buffer to which 5% glycerol was added
Summary
Leishmania donovani salvage all pyrimidines through uracil phosphoribosyltransferase (LdUPRT). Genetic ablation of either carbamoyl phosphate synthetase (CPS), the first enzyme of pyrimidine biosynthesis, or UMPS in L. donovani confer pyrimidine auxotrophy that can be circumvented by supplementation of the defined growth medium with uracil, uridine, deoxyuridine, cytidine, or deoxycytidine [3, 5] Both the ⌬cps and the ⌬umps null mutants exhibit a striking collateral supersensitivity to uracil, which is innocuous to wild type parasites, that is not observed with any of the ribonucleosides [5]. Despite the pyrimidine auxotrophy observed for ⌬cps and ⌬umps L. donovani promastigotes in culture, both knock-out lines sustain relatively robust infections in mice (Ref. 5 and data not shown) These findings imply that the null mutants within the macrophage phagolysosome can access a source of host pyrimidines that can satisfy the pyrimidine nucleotide requirements of the parasite. It is conjectured that this substrate inhibition of LdUPRT by uracil affords the parasite a protective mechanism to protect its nutritionally indispensable purine salvage mechanism and to maintain an equilibrium between purine and pyrimidine nucleotide pools in the parasite
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