A novel calcium-dependent soluble inorganic pyrophosphatase from the trypanosomatid Leishmania major.
A single-copy gene IPP encoding a putative soluble inorganic pyrophosphatase (LmsPPase, EC 3.6.1.1) was identified in the genome of the parasite protozoan Leishmania major. The full-length coding sequence (ca. 0.8 kb) was obtained from genomic DNA by polymerase chain reaction (PCR) and cloned into an Escherichia coli expression vector, and was overexpressed for functional protein purification and characterization. The recombinant LmsPPase, purified to electrophoretic homogeneity by a two-step chromatography procedure, exhibited a predicted molecular mass of ca. 30 kDa. The enzyme has an absolute requirement for divalent cations, exhibits a pH optimum of 7.5-8.0 and does not hydrolyze polyphosphates or adenosine triphosphate (ATP). LmsPPase differs from previously studied soluble pyrophosphatases with respect to cation selectivity, Ca(2+) being far more effective than Mg(2+). Comparisons to known sPPases show a short N-terminal extension predicted to be a mitochondrial transit peptide, and changes in active-site residues and the neighboring region. Subcellular fractionation of L. major promastigotes suggests a mitochondrial localization. Molecular phylogenetic analysis indicates that LmsPPase is a highly divergent eukaryotic Family I sPPase, perhaps an ancestral class of eukaryotic sPPases functionally adapted to a calcium-rich, probably mitochondrial, environment.
Gómez-García
María R
MR
Instituto de Parasitología y Biomedicina López-Neyra, CSIC, c/Ventanilla n degrees 11, 18001 Granada, Spain.
Ruiz-Pérez
Luis M
LM
González-Pacanowska
Dolores
D
Serrano
Aurelio
A
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000201:enzymology; D018503:Genome, Protozoan; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity
D011994:Recombinant Proteins; D043564:Inorganic Pyrophosphatase; D002118:Calcium