Molecular characterization of phosphoribosylpyrophosphate synthetase from Leishmania donovani.
The phosphoribosylpyrophosphate synthetase (PRS) enzyme from parasitic protozoa plays a critical role in the acquisition of exogenous purine bases by providing the phosphoribosylpyrophosphate substrate for phosphoribosylation. To characterize a PRS enzyme from parasitic protozoa, the prs gene was isolated from a genomic library of Leishmania donovani DNA. A 1936-bp SalI fragment was sequenced that encompassed an open reading frame of 1113 nucleotides encoding a polypeptide of 371 amino acids and 40 787 Da. After gap alignment, the leishmanial PRS exhibited 40-42% amino acid identity with a variety of mammalian and prokaryotic PRSs. L. donovani PRS also contained an approx. 20-amino acid stretch that was highly homologous to the phosphoribosylpyrophosphate binding domains of mammalian phosphoribosyltransferase enzymes. Two prs-specific transcripts of 2.6 and 2.1 kb were detected by Northern analysis, and Southern blots of genomic DNA implied that the prs locus was not tandemly repeated in the L. donovani genome. PRS activity was detected in L. donovani extracts, and apparent Km values of approx. 30 microM and approx. 1 mM were calculated for ribose-5-phosphate and ATP, respectively. PRS was sensitive to inhibition by AMP and ADP but refractory to IMP, GMP, GTP, CTP, and UTP. The high apparent Km value of the parasite enzyme for ATP and its insensitivity to inhibition by many nucleotides suggested that kinetic differences between the L. donovani and human PRSs could provide an avenue for rational therapeutic manipulation of parasitic disease. The isolation of the L. donovani prs gene now provides an opportunity to genetically dissect the determinants responsible for the function and regulation of this indispensable enzyme of purine and pyrimidine metabolism in a genus of parasitic protozoa.
Hendrickson
N
N
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
Allen
T
T
Ullman
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D051381:Rats; D015183:Restriction Mapping; D012268:Ribose-Phosphate Pyrophosphokinase / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D016054:DNA, Protozoan; D012268:Ribose-Phosphate Pyrophosphokinase