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.