title: 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 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.
authors.affiliation: Centro Universitário Luterano de Palmas, Universidade Luterana do Brasil, CEULP/ULBRA Palmas, TO, Brazil.
authors.forename: Edson Roberto
authors.initials: ER
authors.lastname: da Silva
authors.forename: Maria Fernanda Laranjeira
authors.initials: MF
authors.lastname: da Silva
authors.forename: Hannes
authors.initials: H
authors.lastname: Fischer
authors.forename: Renato A
authors.initials: RA
authors.lastname: Mortara
authors.forename: Mario Gustavo
authors.initials: MG
authors.lastname: Mayer
authors.forename: Karine
authors.initials: K
authors.lastname: Framesqui
authors.forename: Ariel Mariano
authors.initials: AM
authors.lastname: Silber
authors.forename: Lucile Maria
authors.initials: LM
authors.lastname: Floeter-Winter
mesh_terms: D000818:Animals; D001119:Arginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D007891:Leishmania / Q000737:chemistry* / Q000201:enzymology*; D008830:Microbodies / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009952:Ornithine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D014508:Urea / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014508:Urea; D001120:Arginine; D009952:Ornithine; D001119:Arginase