Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani.
The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.
Yang
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Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
Borchardt
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D043383:Adenosylhomocysteinase; D000818:Animals; D000977:Antiparasitic Agents / Q000494:pharmacology; D002474:Cell-Free System; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000977:Antiparasitic Agents; D011994:Recombinant Proteins; D006867:Hydrolases; D043383:Adenosylhomocysteinase