title: GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites.
abstract: Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Mail Code L224, Portland, OR 97239, USA.
authors.forename: Jan M
authors.initials: JM
authors.lastname: Boitz
authors.affiliation: Institute of Parasitology and Centre for Host-Parasite Interactions, Macdonald Campus of McGill University, 21 111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, H9X3V9, Canada.
authors.forename: Armando
authors.initials: A
authors.lastname: Jardim
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Mail Code L224, Portland, OR 97239, USA. Electronic address: ullmanb@ohsu.edu.
authors.forename: Buddy
authors.initials: B
authors.lastname: Ullman
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism*; D050899:GMP Reductase / Q000235:genetics* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005838:Genotype; D006157:Guanosine Monophosphate / Q000378:metabolism*; D007168:IMP Dehydrogenase / Q000235:genetics / Q000378:metabolism; D007893:Leishmania donovani / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D021381:Protein Transport; D011687:Purines / Q000378:metabolism; D034622:RNA Interference
chemical_list: D011687:Purines; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D007168:IMP Dehydrogenase; D050899:GMP Reductase; C030985:purine
keywords: Adenylate; Guanylate; Leishmania; Purine; Salvage; Uncoupling