Methionine aminopeptidase 2 (MAP2) is a principal regulator of apoptosis for Leishmania donovani and a potential candidate for the design and synthesis of novel antileishmanials. The LdMAP2 gene was cloned in pET28a(+)-SUMO vector, expressed in E. coli and then purified by chromatographic methods. It was found to be a monomer and required divalent metal ion for its activity against synthetic substrates with Co(II), Mg(II), Mn(II) and Ni(II) being the major activators. Moreover, Ca(II) showed the tightest binding with Km value of 124.7 ± 9.2 μM, while Co(II) proved most efficient for catalysis with kcat value of 128.1 ± 4 min-1. The naturally occurring aminopeptidase B inhibitor bestatin was found to be a potent inhibitor of LdMAP2 with a Ki value of 0.86 μM. Further, structural studies with circular dichroism (CD) showed an increase in the α-helical and β-sheet contents and a decrease in random coils in LdMAP2 upon interactions with both bestatin and fluorogenic substrates. Finally, structural studies pointed out key differences in the structure of LdMAP2 and HsMAP2 and their interactions with inhibitor bestatin, Ala-AMC, Leu-AMC and Met-AMC. The structural differences of two orthologs and different binding modes with bestatin can be crucial for the development of novel and specific inhibitor against leishmaniasis.
Keywords: Bestatin; Enzyme kinetics; Leishmania donovani; Methionine aminopeptidase 2; Molecular docking; Molecular dynamics studies.
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