Cloning of the gene encoding Leishmania donovani S-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy.
A full-length gene encoding the S-adenosylhomocysteine hydrolase (AdoHcyase) enzyme has been isolated from a genomic library of Leishmania donovani DNA in lambda GEM-11 by cross-hybridization to the full-length human AdoHcyase cDNA. The nucleotide sequence of the SalI fragment contained a single open reading frame that encoded a polypeptide of 438 amino acids (47,712 Da). After maximum gap alignment, the predicted amino acid sequence of the leishmanial AdoHcyase was 70-73% identical to AdoHCyases from higher eukaryotes. In addition, a data base search revealed that the primary structure of all AdoHcyase proteins was highly homologous to that of a protein encoded by a mRNA from Drosophila melanogaster that maps near the r element function of the Abd-b homeotic gene. In Northern blots, the SalI fragment hybridized to a 3.0-kb transcript that presumably encodes the parasite enzyme. Southern blot analysis of genomic DNA revealed that the AdoHcyase gene did not exist as a tandemly repeated array within the L. donovani genome. Moreover, monoclonal antibodies generated against human AdoHcyase recognized a leishmanial protein on immunoblots. Finally, the growth of L. donovani promastigotes could be arrested by micromolar concentrations of 3-deazaaristeromycin (C3Ari) and 9-(trans-2',trans-3'-dihydroxycyclopentanyl)adenine, 2 known inhibitors of mammalian AdoHcyase. C3Ari also induced a substantial expansion of the intracellular pools of both AdoHcy and S-adenosylmethionine (AdoMet), as well as a significant diminution of the AdoMet/AdoHcy ratio. Thus, AdoHcyase may have therapeutic potential for the selective treatment of diseases of parasitic origin.
Henderson
D M
DM
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
Hanson
S
S
Allen
T
T
Wilson
K
K
Coulter-Karis
D E
DE
Greenberg
M L
ML
Hershfield
M S
MS
Ullman
B
B
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics*; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D015183:Restriction Mapping
D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; C054757:9-(2',3'-dihydroxycyclopent-4'-enyl)adenine; C034583:3-deazaaristeromycin; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine