Genetic analysis of spermidine synthase from Leishmania donovani

Mol Biochem Parasitol. 2001 Jul;115(2):217-26. doi: 10.1016/s0166-6851(01)00293-6.

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 Deltaspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Deltaspdsyn 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Culture Media
  • DNA, Protozoan / analysis
  • DNA, Protozoan / genetics
  • Gene Deletion
  • Genes, Protozoan
  • Immunoblotting
  • Leishmania donovani / enzymology*
  • Leishmania donovani / genetics
  • Leishmania donovani / growth & development
  • Molecular Sequence Data
  • Polyamines / metabolism
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Spermidine / metabolism
  • Spermidine Synthase / chemistry
  • Spermidine Synthase / genetics*
  • Spermidine Synthase / isolation & purification
  • Spermidine Synthase / metabolism

Substances

  • Culture Media
  • DNA, Protozoan
  • Polyamines
  • Spermidine Synthase
  • Spermidine

Associated data

  • GENBANK/AF298195