Xanthine phosphoribosyltransferase from Leishmania donovani -: Molecular cloning, biochemical characterization, and genetic analysis

Xanthine phosphoribosyltransferase (XPRT) from Leishmania donovani is a unique enzyme that lacks a mammalian counterpart and is, therefore, a potential target for antiparasitic therapy. To investigate the enzyme at the molecular and biochemical level, a cDNA encoding the L, donovani XPRT was isolated by functional complementation of a purine auxotroph of Escherichia coli that also harbors deficiencies in the prokaryotic phosphoribosyltransferase (PRT) activities. The cDNA was then used to isolate the XPRT genomic clone. XPRT encodes a 241-amino acid protein exhibiting similar to 33% amino acid identity with the L. donovani hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and significant homology with other HGPRT family members. Southern blot analysis revealed that XPRT was a single copy gene that co-localized with HGPRT within a 4.3-kilobase pair (kb) EcoRI fragment, implying that the two genes arose as a result of an ancestral duplication event. Sequencing of this EcoRI fragment confirmed that HGPRT and XPRT were organized in a head-to-tail arrangement separated by an similar to 2.2-kb intergenic region, Both the 3.2-kb XPRT mRNA and XPRT enzyme were significantly up-regulated in Delta hgprt and Delta hgprt/Delta aprt L. donovani mutants. Genetic obliteration of the XPRT locus by targeted gene replacement indicated that XPRT was not an essential gene under most conditions and that the Delta xprt null strain was competent of salvaging all purines except xanthine, XPRT was overexpressed in E, coli and the recombinant protein purified to homogeneity, Kinetic analysis revealed that the XPRT preferentially phosphoribosylated xanthine but could also recognize hypoxanthine and guanine, K-m values of 7.1, 448.0, and >100 mu M and k(cat) values of 3.5, 2.6, and similar to 0.003 s(-1) were calculated for xanthine, hypoxanthine, and guanine, respectively. The XPRT gene and XPRT protein provide the requisite molecular and biochemical reagents for subsequent studies to validate XPRT as a potential therapeutic target.