Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania d onovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purifie d from cell-free extracts of E. coli using three chromatographic steps (DEA E-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharo se ion exchange). The purified recombinant L. donovani enzyme exists as a t etramer with a molecular weight of similar to 48 kDa for each subunit. Unli ke recombinant human AdoHcy hydrolase, the catalytic activity of the recomb inant L. donovani enzyme was shown to be dependent on the concentration of NAD(+) in the incubation medium. The dissociation constant (K-d) for NAD(+) with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 muM. The K-m v alues for the natural substrates of the enzyme, AdoHcy, Ado, and Hey, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 muM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory e ffects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors differ ent than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic e nzyme as potential antiparasitic agents, (C) 2000 Academic Press.