Molecular characterization of recombinant arginase of Leishmania donovani.

Arginase catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and hence potential drug target for the treatment of leishmaniasis. The arginase from Leishmania donovani (LdARG) was cloned, overexpressed and characterized. Analysis of the deduced amino acid sequence of LdARG with homologous enzyme from other trypanosomatids arginases identified a non-conserved 12 residues long segment VWGLIERTFLSA from position 161-172. This counter segment in L. mexicana arginase exhibits a different conformation compared with human arginase I. The pH and temperature optima of LdARG were 9.0 and 37 °C, respectively. Biochemical studies revealed that the K for the substrate L-arginine was 24.76 ± 0.06 mM. Molecular modeling of LdARG studies revealed that the glutamic acid residue at position 288 plays a role in substrate binding. The importance of this glutamic acid residue was validated by constructing a mutant variant of LdARG (E288Q-LdARG) by replacing glutamic acid with glutamine through site-directed mutagenesis. The K value of mutant variant for L-arginine was found to be 107 ± 0.18 mM. The increase in K value of E288Q-LdARG as compared to LdARG suggested that substrate binding was significantly affected which could be exploited further. Studies on biochemical and structural characterization of recombinant LdARG will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.