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

Xanthine phosphoribosyltransferase (XPRT) from Leishmania donovani is a uni que enzyme that lacks a mammalian counterpart and is, therefore, a potentia l target for antiparasitic therapy. To investigate the enzyme at the molecu lar and biochemical level, a cDNA encoding the L, donovani XPRT was isolate d by functional complementation of a purine auxotroph of Escherichia coli t hat 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 phosphoribosyltransfera se (HGPRT) and significant homology with other HGPRT family members. Southe rn blot analysis revealed that XPRT was a single copy gene that co-localize d 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. Sequenci ng 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 regi on, Both the 3.2-kb XPRT mRNA and XPRT enzyme were significantly up-regulat ed in Delta hgprt and Delta hgprt/Delta aprt L. donovani mutants. Genetic o bliteration of the XPRT locus by targeted gene replacement indicated that X PRT was not an essential gene under most conditions and that the Delta xprt null strain was competent of salvaging all purines except xanthine, XPRT w as overexpressed in E, coli and the recombinant protein purified to homogen eity, Kinetic analysis revealed that the XPRT preferentially phosphoribosyl ated 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, respe ctively. The XPRT gene and XPRT protein provide the requisite molecular and biochemical reagents for subsequent studies to validate XPRT as a potentia l therapeutic target.