Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

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 K m values of 20 and 99 μm for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent K m 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. Background: Leishmania donovani salvage all pyrimidines through uracil phosphoribosyltransferase (LdUPRT). Results: LdUPRT phosphoribosylates uracil, 5-fluorouracil, and 4-thiouracil and is susceptible to substrate inhibition. Conclusion: LdUPRT recognizes pyrimidine analogs, and substrate inhibition by LdUPRT explains the supersensitivity of pyrimidine auxotrophs to uracil. Significance: Substrate inhibition of LdUPRT provides a mechanism for uracil susceptibility and offers a protective function for the parasite.