S-adenosylmethionine decarboxylase from Leishmania donovani: Molecular, genetic, and biochemical characterization of null mutants and overproducers

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Δadometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Δadometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Δadometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Δadometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Δadometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5′-(((Z)-4-amino-2-butenyl)methylamino)-5′-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methyl-glyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.