Step In Phospholipid Biosynthesis Research Articles

In Meso Crystallization of the Integral Membrane Glycerol 3-Phosphate Acyltransferase with Substrates

Here we retrospectively report the successful application of the “host lipid screening” strategy in the in meso crystallization of PlsY, a bacterial glycerol 3-phosphate (G3P) acyltransferase that catalyzes the committed step in phospholipid biosynthesis. Two monoacylglycerols (MAGs) with different chain lengths, namely, the 9.9 MAG (monoolein, nine carbons on either side of the cis-double bond in the acyl chain) and the 7.8 MAG, were the two main host lipids used in this study. The native PlsY crystals growing in 9.9 MAG diffracted to 1.62 A. However, the selenomethioninyl crystals growing in the same host lipid under similar precipitant conditions diffracted poorly (4.3 A). Switching the host lipid to 7.8 MAG dramatically improved crystal quality in both size and diffraction, yielding a 2.0-A data set, with which the structure was solved. Along with low-temperature crystallization, 7.8 MAG was also critical for cocrystallization of PlsY with the unstable lipid substrate, acyl phosphate (acylP). Interest...

Molecular drivers of mitochondrial membrane proliferation in response to cold acclimation in threespine stickleback

Little is known about how the synthesis of mitochondrial phospholipids is integrated into mitochondrial biogenesis in fish or mammals. Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the addition of fatty acyl CoA to the sn-1 position of glycerol-3-phosphate, in what is considered the rate-limiting step in phospholipid biosynthesis. Previous studies have shown that mitochondrial volume density increases in oxidative skeletal muscle but not liver of Gasterosteus aculeatus (threespine stickleback) in response to cold acclimation. We hypothesized that maximal activity of GPAT would increase in oxidative skeletal muscle but not liver during cold acclimation, coinciding with mitochondrial biogenesis. GPAT activity was measured in liver and oxidative skeletal (pectoral adductor) muscle of threespine stickleback acclimated to 8°C or 20°C. In addition, mRNA levels of enzymes involved in phospholipid synthesis, including cytidine diphosphodiacylglycerol synthase-1 (CDS1), CDS2, GPAT1, GPAT2 and 1-acylglycerol 3-phosphate acyltransferase-2 (AGPAT2), were quantified in liver and pectoral muscle of stickleback harvested during cold acclimation. GPAT activity and transcript levels of AGPAT2 increased in response to cold acclimation in pectoral muscle but not liver. Transcript levels of GPAT1 increased in liver but not pectoral muscle. Overall our results suggest that the activity of GPAT, and possibly AGPAT as well, increase during cold acclimation and may contribute to mitochondrial phospholipid biosynthesis required for mitochondrial biogenesis.

511 Potentiation of cell sensitivity to the DNA topoisomerase I inhibitor gimatecan by TRAIL in prostate carcinoma cells

This chapter discusses the biosynthesis of phospholipids, which consists of (1) the basic pathways leading to de novo formation of phospholipid and (2) special transformations that interconvert one phospholipid to another. There is more than one way that nature has developed to make specific phospholipids, and often, different species or subcellular organelles have developed different approaches. For example, phosphatidylserine is made in prokaryotes, such as Escherichia coli by the reaction of L-serine with CDPdiglyceride, a de novo step, whereas mammalian cells, such as rat liver utilize an L-serine-phosphatidylethanolamine exchange enzyme in a special transformation or transesterification of a preformed phospholipid not requiring the expenditure of a high-energy phosphate. The importance and discovery of cytidine nucleotides are discussed in the chapter. Cytidine nucleotides are responsible for the de novo step in phospholipid biosynthesis.

The Plasmodium falciparum PfGatp is an endoplasmic reticulum membrane protein important for the initial step of malarial glycerolipid synthesis.

During its 48-h asexual life cycle within human erythrocytes, Plasmodium falciparum grows to many times its own size and divides to produce 16-32 new parasites. This rapid multiplication requires active synthesis of new membranes and is fueled by phospholipid precursors and fatty acids that are scavenged from the human host. Plasmodium membrane biogenesis relies heavily on the expression of parasite enzymes that incorporate these precursors into phospholipids. However, little is known about the genes involved in membrane biogenesis or where this process takes place within the parasite. Here, we describe the analysis in P. falciparum of the first step of phospholipid biosynthesis that controls acylation of glycerol 3-phosphate (GPAT) at the sn-1 position. We show that this activity is of parasite origin and is specific for glycerol 3-phosphate substrate. We have identified the gene, PfGAT, encoding this activity in P. falciparum and reconstituted its codon composition for optimal expression in the yeast Saccharomyces cerevisiae. PfGAT complements the lethality of a yeast double mutant gat1Deltagat2Delta, lacking GPAT activity. Biochemical analysis revealed that PfGatp is a low affinity GPAT enzyme with a high specificity for C16:0 and C16:1 substrates. PfGatp is an integral membrane protein of the endoplasmic reticulum expressed throughout the intraerythrocytic life cycle of the parasite but induced mainly at the trophozoite stage. This study, which describes the first protozoan GPAT gene, reveals an important role for the endoplasmic reticulum in the initial step of Plasmodium membrane biogenesis.

Inhibition of acetyl coenzyme A carboxylase activity restores expression of the INO1 gene in a snf1 mutant strain of Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1Delta strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the beta subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1Delta strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1Delta strains. Together, these findings indicate that the inositol auxotrophy of snf1Delta strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

Multiple lysophosphatidic acid acyltransferases in Neisseria meningitidis.

Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are critical phospholipid intermediates in the biosynthesis of cell membranes. In Escherichia coli, LPA acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase; EC 2.3.1.51) catalyses the transfer of an acyl chain from either acyl-coenzyme A or acyl-acyl carrier protein onto LPA to produce PA. While E. coli possesses one essential LPA acyltransferase (PlsC), Neisseria meningitidis possesses at least two LPA acyltransferases. This study describes the identification and characterization of nlaB (neisserial LPA acyltransferase B), the second LPA acyltransferase identified in N. meningitidis. The gene was located downstream of the Tn916 insertion in N. meningitidis mutant 469 and differed in nucleotide and predicted amino acid sequence from the previously characterized neisserial LPA acyltransferase homologue nlaA. NlaB has specific LPA acyltransferase activity, as demonstrated by complementation of an E. coli plsC(Ts) mutant in trans, by decreased levels of LPA acyltransferase activity in nlaB mutants and by lack of complementation of E. coli plsB26,X50, a mutant defective in the first acyltransferase step in phospholipid biosynthesis. Meningococcal nlaA mutants accumulated LPA and demonstrated alterations in membrane phospholipid composition, yet retained LPA acyltransferase activity. In contrast, meningococcal nlaB mutants exhibited decreased LPA acyltransferase activity, but did not accumulate LPA or display any other observable membrane changes. We propose that N. meningitidis possesses at least two LPA acyltransferases to provide for the production of a greater diversity of membrane phospholipids.

Galactosemic Cataractogenesis Disrupts Intracellular Interactions and Changes the Substrate Specificity of Choline/Ethanolamine Kinase

The functional characteristics of enzymes depends upon their environment, and within physiologically intact cells, many metabolic pathways are thought to involve multienzyme complexes and other enzyme–enzyme interactions that increase efficiency and specificity by mechanisms such as channeling of intermediates. A disease such as cataract may change the intracellular environment, but the effects of these changes on enzyme–enzyme interactions can be observed only in relatively intact cells, and in enzymes that have unambiguously different properties in different environments. In intact rat lenses, choline and ethanolamine are phosphorylated independently, with no competition between the two compounds, as the first step of phospholipid biosynthesis. However, disruption of lens structure and intracellular interactions by homogenization leads to a paradoxical change in enzymic properties, causing choline and ethanolamine to become competing alternative substrates of a single enzyme that resembles the purified choline/ethanolamine kinase from liver and other tissues. The properties of ethanolamine kinase in intact cataractous lenses from rats fed a 50% galactose diet for 7–14 days were intermediate between those of intact control lenses and those in lens homogenates. In monolayers of human and dog lens epithelial cells and human retinal pigment epithelial cells ethanolamine kinase was similar to that in intact tissue, showing that the kinetic differences between intact lenses and homogenates result from the intracellular environment, not from artifacts of diffusion, and that they are not exclusive to rats or to lens cells. Results with intact lenses from monkeys, rabbits, pigs and dogs showed some differences between species, but in every case, choline had little or no effect on the phosphorylation of radiolabeled ethanolamine. Further studies will be necessary to determine how the changes in intracellular environment during cataractogenesis affect other enzymes and whether other model systems for cataractogenesis cause similar changes.

1] The biosynthesis of phospholipids

Publisher Summary This chapter discusses the biosynthesis of phospholipids, which consists of (1) the basic pathways leading to de novo formation of phospholipid and (2) special transformations that interconvert one phospholipid to another. There is more than one way that nature has developed to make specific phospholipids, and often, different species or subcellular organelles have developed different approaches. For example, phosphatidylserine is made in prokaryotes, such as Escherichia coli by the reaction of L-serine with CDPdiglyceride, a de novo step, whereas mammalian cells, such as rat liver utilize an L-serine-phosphatidylethanolamine exchange enzyme in a special transformation or transesterification of a preformed phospholipid not requiring the expenditure of a high-energy phosphate. The importance and discovery of cytidine nucleotides are discussed in the chapter. Cytidine nucleotides are responsible for the de novo step in phospholipid biosynthesis.

Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis: Genetic analysis of glycerol-3-phosphate acyltransferase mutants

Escherichia coli mutants possessing temperature-sensitive lesions in glycerol-3-phosphate acyltransferase, the enzyme catalysing the first step in phospholipid biosynthesis, have been characterized genetically. By recombinational and complementation tests, the mutants have been found to map in a single locus, called plsA, which is cotransducibile with the purE locus and lies between the purE and proC loci at minute 13 on the E. coli genetic map.