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Genetic analysis of spermidine synthase fromLeishmania donovani

Authors
Journal
Molecular and Biochemical Parasitology
0166-6851
Publisher
Elsevier
Publication Date
Volume
115
Issue
2
Identifiers
DOI: 10.1016/s0166-6851(01)00293-6
Keywords
  • Leishmania Donovani
  • Spermidine Synthase
  • Gene Knockouts
  • Polyamines
Disciplines
  • Biology
  • Chemistry
  • Design
  • Medicine

Abstract

Abstract The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Δ spdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Δ spdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3–4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.

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