Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum.
In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum-infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 +/- 19 microM and a VMAX of 680 +/- 49 nmol of phosphatidylethanolamine formed h-1 mg-1 protein. Site-directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits (alpha and beta) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.
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Dynamique Moléculaire des Interactions Membranaires, CNRS UMR 5539, cc107, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France.
Eldin
Patrick
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Cathiard
Anne-Marie
AM
Vial
Henri
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004912:Erythrocytes / Q000469:parasitology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase