Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

ABSTRACT

Arginase (l-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysisof l-arginine to l-ornithine and urea. In Leishmania spp., the biological role of the enzyme may beinvolved in modulating NO production upon macrophage infection. Previously, we cloned and characterizedthe arginase gene from Leishmania (Leishmania) amazonensis. In the presentwork,we successfullyexpressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterizationof both the native and recombinant enzymes. We obtained KM and Vmax values of 23.9(±0.96)mM and192.3 mol/minmg protein (±14.3), respectively, for the native enzyme. For the recombinant counterpart,KM was 21.5(±0.90)mMand Vmax was 144.9(±8.9) mol/min mg. Antibody against the recombinantprotein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data fromlight scatteringand small angle X-ray scattering showed that a trimeric state is the active form of the protein.Wedetermined empirically that a manganesewash at room temperature is the best condition to purify activeenzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirmthe structural disposition of histidine at positions 3 and 324. The determined structural parametersprovide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase,which could subsequently point to a candidate for leishmaniasis therapy.