Publications

• From protease to decarboxylase: the molecular metamorphosis of phosphatidylserine decarboxylase.

  Choi J.Y., Duraisingh M.T., Marti M., Ben Mamoun C., Voelker D.R.

  Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is forme ... >> More

  Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large β-subunit, and a smaller α-subunit, which harbors the prosthetic group at its N terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFXRX6RX12PXD motif, two uniquely conserved histidine residues within a PXXYHXXHXP motif, and a uniquely conserved serine residue within a GS(S/T) motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site-directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that Asp-139, His-198, and Ser-308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GS(S/T) motif found in all PSDs, the Gly-307 residue is also essential, but the Ser/Thr-309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group. << Less

  J. Biol. Chem. 290:10972-10980(2015) [PubMed] [EuropePMC]

• METABOLISM AND FUNCTION OF BACTERIAL LIPIDS. II. BIOSYNTHESIS OF PHOSPHOLIPIDS IN ESCHERICHIA COLI.

  KANFER J., KENNEDY E.P.

  J Biol Chem 239:1720-1726(1964) [PubMed] [EuropePMC]

• Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum.

  Baunaure F., Eldin P., Cathiard A.M., Vial H.

  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 ... >> More

  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. << Less

  Mol. Microbiol. 51:33-46(2004) [PubMed] [EuropePMC]

• Identification of a non-mitochondrial phosphatidylserine decarboxylase activity (PSD2) in the yeast Saccharomyces cerevisiae.

  Trotter P.J., Voelker D.R.

  Phosphatidylserine decarboxylase (PSD1) plays a central role in the biosynthesis of aminophospholipids in both prokaryotes and eukaryotes by catalyzing the synthesis of phosphatidylethanolamine. Recent reports (Trotter, P. J., Pedretti, J., and Voelker, D. R. (1993) J. Biol. Chem. 268, 21416-21424 ... >> More

  Phosphatidylserine decarboxylase (PSD1) plays a central role in the biosynthesis of aminophospholipids in both prokaryotes and eukaryotes by catalyzing the synthesis of phosphatidylethanolamine. Recent reports (Trotter, P. J., Pedretti, J., and Voelker, D. R. (1993) J. Biol. Chem. 268, 21416-21424; Clancey, C. J., Chang, S.-C., and Dowhan, W. (1993) J. Biol. Chem. 268, 24580-24590) described the cloning of a yeast structural gene for this enzyme (PSD1) and the creation of the null allele. Based on the phenotype of strains containing a null allele for PSD1 (psd1-delta 1::TRP1) it was hypothesized that yeast have a second phosphatidylserine decarboxylase. The present studies demonstrate the presence of a second enzyme activity (denoted PSD2), which, depending on the method of evaluation, accounts for 4-12% of the total cellular phosphatidylserine decarboxylase activity found in wild type. Recessive mutations resulting in loss of this enzyme activity (denoted psd2) in cells containing the psd1-delta 1::TRP1 null allele also result in ethanolamine auxotrophy. When incubated with [3H]serine these double mutants accumulate label in phosphatidylserine, while very little (< 5%) is converted to phosphatidylethanolamine. In addition, these mutants have a approximately 70% decrease in the amount of total phosphatidylethanolamine even when grown in the presence of exogenous ethanolamine. Strains containing psd1 or psd2 mutations were utilized for the subcellular localization of the PSD2 enzyme activity. Unlike the PSD1 activity, the PSD2 enzyme activity does not localize to the mitochondria, but to a low density subcellular compartment with fractionation properties similar to both vacuoles and Golgi. << Less

  J. Biol. Chem. 270:6062-6070(1995) [PubMed] [EuropePMC]

• Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

  Satre M., Kennedy E.P.

  Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum ... >> More

  Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase. << Less

  J. Biol. Chem. 253:479-483(1978) [PubMed] [EuropePMC]

• Coordinate regulation of phosphatidylserine decarboxylase activity and phospholipid N-methylation in yeast.

  Carson M.A., Emala M., Hogsten P., Waechter C.J.

  Membranes isolated from Saccharomyces cerevisiae, strain ATCC 26615, catalyze the decarboxylation of exogenous phosphatidylserine added as an aqueous dispersion in detergent. Active preparations of the decarboxylase can be obtained by extracting salt-washed membranes with 0.5% Cutscum . The proper ... >> More

  Membranes isolated from Saccharomyces cerevisiae, strain ATCC 26615, catalyze the decarboxylation of exogenous phosphatidylserine added as an aqueous dispersion in detergent. Active preparations of the decarboxylase can be obtained by extracting salt-washed membranes with 0.5% Cutscum . The properties of the phosphatidylserine decarboxylase activity associated with a particulate fraction and the detergent extracts have been characterized by assaying the enzymatic conversion of exogenous [14C]phosphatidylserine to [14C]phosphatidylethanolamine. The yeast decarboxylase does not require a divalent cation and is inhibited by hydroxylamine and p-hydroxymercuribenzoate. The rate of decarboxylation of exogenous phosphatidylserine catalyzed by membranes prepared from cells grown in the presence of choline is reduced by approximately 60% compared to membranes from cells grown in a choline-deficient medium. Relatively smaller reductions in phosphatidylserine decarboxylase activity are also seen in cells grown in the presence of mono- or dimethylethanolamine. In vitro incorporation studies with [14C]serine demonstrate that endogenous, prelabeled phosphatidylserine can be utilized for the biosynthesis of phosphatidylcholine by the coupled action of the hydroxylamine-sensitive decarboxylase and the phospholipid N-methyltransferases in the presence of 2 mM S-adenosylmethionine. A similar comparative enzymatic study shows that the rates of synthesis and decarboxylation of [14C]phosphatidylserine, as well as phospholipid N-methylation, are lower for membranes prepared from cells grown in the presence of choline relative to identical preparations from cells grown in the absence of choline. These studies describe the properties of particulate and detergent-solubilized phosphatidylserine decarboxylase activity in S. cerevisiae and provide evidence that its activity is regulated in coordination with other enzymes in the pathway for phosphatidylcholine biosynthesis involving N-methylation. << Less

  J. Biol. Chem. 259:6267-6273(1984) [PubMed] [EuropePMC]

• Purification and properties of phosphatidylserine decarboxylase from Escherichia coli.

  Dowhan W., Wickner W.T., Kennedy E.P.

  J. Biol. Chem. 249:3079-3084(1974) [PubMed] [EuropePMC]

• Cloning, sequencing, and disruption of the Bacillus subtilis psd gene coding for phosphatidylserine decarboxylase.

  Matsumoto K., Okada M., Horikoshi Y., Matsuzaki H., Kishi T., Itaya M., Shibuya I.

  The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of pss, the structural gene for phosphatidylserine synthase t ... >> More

  The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of pss, the structural gene for phosphatidylserine synthase that catalyzes the preceding reaction in phosphatidylethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456-7461, 1994). Introduction of a plasmid containing the psd gene into temperature-sensitive Escherichia coli psd-2 mutant cells allowed growth at otherwise restrictive temperature. Phosphatidylserine was not detected in the psd-2 mutant cells harboring the plasmid; it accumulated in the mutant up to 29% of the total phospholipids without the plasmid. An enzyme activity that catalyzes decarboxylation of 14C-labeled phosphatidylserine to form phosphatidylethanolamine was detected in E. coli psd-2 cells harboring a Bacillus psd plasmid. E. coli cells harboring the psd plasmid, the expression of which was under the control of the T7phi10 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The psd gene, together with pss, was located by Southern hybridization to the 238-to 306-kb SfiI-NotI fragment of the chromosome. A B. subtilis strain harboring an interrupted psd allele, psd1::neo, was constructed. The null psd mutant contained no phosphatidylethanolamine and accumulated phosphatidylserine. It grew well without supplementation of divalent cations which are essential for the E. coli pssA null mutant lacking phosphatidylethanolamine. In both the B. subtilis null pss and psd mutants, glucosyldiacylglycerol content increased two-to fourfold. The results suggest that the lack of phosphatidylethanolamine in the B. subtilis membrane may be compensated for by the increases in the contents of glucosyldiacylglycerols by an unknown mechanism. << Less

  J. Bacteriol. 180:100-106(1998) [PubMed] [EuropePMC]