Related Experiment Videos
Xanthine phosphoribosyltransferase from Leishmania donovani. Molecular cloning, biochemical characterization, and
A Jardim1, S E Bergeson, S Shih
1Department of Biochemistry, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Abstract:
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 approximately 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 approximately 2.2-kb intergenic region. Both the 3.2-kb XPRT mRNA and XPRT enzyme were significantly up-regulated in Deltahgprt and Deltahgprt/Deltaaprt 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 Deltaxprt 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 microM and k(cat) values of 3.5, 2.6, and approximately 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.
Insights
Xanthine phosphoribosyltransferase (XPRT) from Leishmania donovani is a potential antiparasitic target. Researchers isolated and characterized the XPRT gene and protein, revealing its biochemical properties and potential for drug development.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- Leishmania donovani causes leishmaniasis, a neglected tropical disease.
- Xanthine phosphoribosyltransferase (XPRT) is a unique enzyme in L. donovani, absent in mammals, making it a promising therapeutic target.
- Understanding XPRT's molecular and biochemical characteristics is crucial for developing novel antiparasitic strategies.
Purpose of the Study:
- To isolate and characterize the Xanthine phosphoribosyltransferase (XPRT) gene and protein from Leishmania donovani.
- To investigate the biochemical properties and substrate specificity of L. donovani XPRT.
- To assess the potential of XPRT as a target for antiparasitic drug development.
Main Methods:
- Isolation of L. donovani XPRT cDNA by functional complementation in Escherichia coli.
- Genomic cloning and sequencing of the XPRT gene.
- Southern blot analysis to determine gene copy number and organization.
- Gene replacement to create a Deltaxprt null mutant.
- Overexpression and purification of recombinant XPRT protein.
- Kinetic analysis of XPRT activity with various purine substrates.
Main Results:
- The XPRT gene was isolated and found to encode a 241-amino acid protein with homology to hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
- XPRT and HGPRT genes are located adjacent to each other, suggesting an ancestral duplication event.
- The Deltaxprt null strain could salvage all purines except xanthine, indicating XPRT is not essential under most conditions.
- Purified recombinant XPRT preferentially phosphoribosylated xanthine, with lower activity towards hypoxanthine and guanine.
Conclusions:
- The L. donovani XPRT gene and protein have been molecularly and biochemically characterized.
- XPRT exhibits distinct substrate specificity, primarily acting on xanthine.
- The findings provide essential reagents and data to validate XPRT as a viable antiparasitic therapeutic target.