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 Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc 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 Delta adometdc 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 Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc 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 methylglyoxyl 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.
Roberts
Sigrid C
SC
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97201-3098, USA.
Scott
Jerry
J
Gasteier
Judith E
JE
Jiang
Yuqui
Y
Brooks
Benjamin
B
Jardim
Armando
A
Carter
Nicola S
NS
Heby
Olle
O
Ullman
Buddy
B
D012437:Adenosylmethionine Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003839:Deoxyadenosines / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010802:Phylogeny; D011073:Polyamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection; D014349:Trypanosoma cruzi / Q000201:enzymology
D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D011073:Polyamines; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C065859:MDL 73811; D012437:Adenosylmethionine Decarboxylase