Arginase in Leishmania.

The presence of different sets of several enzymes that participate in the Krebs-Henseleit cycle has been used to identify several genera of trypanosomatids. One of these enzymes is arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1), a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. Arginase activity has been detected in Leishmania, Crithidia and Leptomonas but not in Trypanosoma, Herpetomonas or Phytomonas. The ureotelic behavior of some trypanosomatids is not due to urea excretion but to the production of ornithine to supply the polyamine pathway, which is essential for replication. Leishmania is found inside macrophages in the mammalian host and to live in these cells, the parasite must escape from several microbicidal mechanisms, such as nitric oxide (NO) production mediated by inducible nitric oxide synthase (iNOS). Since arginase and iNOS use the L-arginine as substrate, the amount of this amino acid available for both pathways is critical for parasite replication. In both promastigotes and amastigotes, arginase is located in the glycosome indicating that arginine trafficking in the cell is used to provide the optimal concentration of substrate for arginase. Arginine uptake by the parasite is also important in supplying the arginase substrate. Leishmania responds to arginine starvation by increasing the amino acid uptake. In addition to the external supply, the internal L-arginine pool also governs the uptake of this amino acid, and the size of this internal pool is modulated by arginase activity. Thus, arginine uptake and arginase activity are important in establishing and maintaining Leishmania infection.

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis.

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253+/-0.01 mM and a maximum velocity of 0.246+/-0.04 nmol/min per 10(7) cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host.

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

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K(M) and V(max) values of 23.9(+/-0.96) mM and 192.3 micromol/min mg protein (+/-14.3), respectively, for the native enzyme. For the recombinant counterpart, K(M) was 21.5(+/-0.90) mM and V(max) was 144.9(+/-8.9) micromol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy.

Leishmania amazonensis arginase compartmentalization in the glycosome is important for parasite infectivity.

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg(-) L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg(-) mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration.