An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate.
We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg(2+) requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, or p-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 +/- 3.0 mm) and long chain polyP (55.9 +/- 5.6 mm) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.
Rodrigues
Claudia O
CO
Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
Ruiz
Felix A
FA
Vieira
Mauricio
M
Hill
Janet E
JE
Docampo
Roberto
R
D017766:Acid Anhydride Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D010802:Phylogeny; D011122:Polyphosphates / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity
D002413:Cations, Divalent; D011122:Polyphosphates; D011994:Recombinant Proteins; D006493:Heparin; D012964:Sodium; D017766:Acid Anhydride Hydrolases; C020137:exopolyphosphatase; D008274:Magnesium