title: Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani.
abstract: Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (K(m)=33.24 μm, k(cat)=1.3 s(-1), K(i)=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting K(m) values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.
authors: Department of Biotechnology, Indian Institute of Technology Guwahati, Assam-781039, India.
authors: Prakash
authors: P
authors: Saudagar
authors: Vikash Kumar
authors: VK
authors: Dubey
mesh_terms: D019732:Amide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019732:Amide Synthases; C064407:trypanothione synthetase