Synthesis Of Phosphatidylglycerol Research Articles

Preferential phosphatidylglycerol synthesis via phosphorus supply through rRNA degradation in the cyanobacterium, Synechocystis sp. PCC 6803, under phosphate-starved conditions.

Photosynthetic organisms often encounter phosphorus (P) limitation in natural habitats. When faced with P limitation, seed plants degrade nucleic acids and extra-plastid phospholipids to remobilize P, thereby enhancing their internal-P utilization efficiency. Although prokaryotic and eukaryotic photosynthetic organisms decrease the content of phosphatidylglycerol (PG) under P-limited conditions, it remains unclear whether PG is degraded for P remobilization. Moreover, information is limited on internal-P remobilization in photosynthetic microbes. This study investigates internal-P remobilization under P-starvation (-P) conditions in a cyanobacterium, Synechocystis sp. PCC 6803, focusing on PG and nucleic acids. Our results reveal that the PG content increases by more than double in the -P culture, indicating preferential PG synthesis among cellular P compounds. Simultaneously, the faster increases of glycolipids counteract this PG increase, which decreases the PG proportion in total lipids. Two genes, glpD and plsX, contribute to the synthesis of diacylglycerol moieties in glycerolipids, with glpD also responsible for the polar head group synthesis in PG. The mRNA levels of both glpD and plsX are upregulated during -P, which would cause the preferential metabolic flow of their P-containing substrates toward glycerolipid synthesis, particularly PG synthesis. Meanwhile, we find that RNA accounts for 62% of cellular P, and that rRNA species, which makes up the majority of RNA, are degraded under -P conditions to less than 30% of their initial levels. These findings emphasize the importance of PG in -P-acclimating cell growth and the role of rRNA as a significant internal-P source for P remobilization, including preferential PG synthesis.

Molecular exposition of broad-spectrum antibacterial efficacy by p-coumaric acid from an edible mushroom Termitomyces heimii: in vitro and in silico approach

P-Coumaric acid was previously reported to contain antioxidant, antidiabetic, anti-inflammatory, antiplatelet, antiulcer and anticancer activities. Along with these, the present work has been conducted to study the antibacterial activity of p-coumaric acid. It could be used to control broad-spectrum microbiome-based inflammation or in cancer control. HPLC analysis of methanolic extract from the mushroom Termitomyces heimii has exhibited a rich fraction of p-coumaric acid (p-CA), which in the pure form showed significant bactericidal potentials. To predict the molecular interactions associated with the bactericidal mechanism of p-CA, the transmembrane protein sequences of Staphylococcus aureus and Escherichia coli were retrieved from the IMG–JGI database, screened and then aligned using Clustal X2 and PHYLIP 3.69 softwares. The common sequences were subjected to tertiary structure prediction using Phyre2 server, followed by quality assessment through the Ramachandran plot. Next, the 3D molecular structure of p-CA was downloaded from PubChem and docked with the selected tertiary structures using Patchdock and showed higher affinity towards 12 common transmembrane protein structures, amongst which CDP-diacylglycerol–glycerol-3-phosphate 3-phosphatidyl transferase (PgsA) exhibited best docking with p-CA on the basis of ACE value (– 249 kcal/mol). This fact revealed that p-CA can block the normal functioning of membrane-bound enzyme PgsA, consequently leading to the interruption in the synthesis and recycling of an essential membrane component phosphatidylglycerol (PG), resulting in membrane disruption followed by cell lysis. Here, for the first time we reported the molecular insights and fundamental biochemical events underlying the bactericidal action by p-CA, to explore new perspective to combat multidrug-resistant bacteria.

Synthesis of Phosphatidyl Glycerol Containing Unsymmetric Acyl Chains Using H-Phosphonate Methodology.

Naturally occurring phospholipids, such as phosphatidyl glycerol (PG), are gaining interest due to the roles they play in disease mechanisms. To elucidate the metabolism of PG, an optically pure material is required, but this is unfortunately not commercially available. Our previous PG synthesis route utilized phosphoramidite methodology that addressed issues surrounding fatty acid substrate scope and glycerol backbone modifications prior to headgroup phosphorylation, but faltered in the reproducibility of the overall pathway due to purification challenges. Herein, we present a robust pathway to optically pure PG in fewer steps, utilizing H-phosphonates that features a chromatographically friendly and stable triethyl ammonium H-phosphonate salt. Our route is also amendable to the simultaneous installation of different acyl chains, either saturated or unsaturated, on the glycerol backbone.

The toc132toc120 heterozygote mutant of Arabidopsis thaliana accumulates decreased levels of the major chloroplast lipids

We used ESI-MS/MS to profile glycerolipids in a mutant of Arabidopsis thaliana that is null and heterozygous for the TOC132 and TOC120 genes, and is referred to as the toc132toc120± mutant. The goal was to assess the impact of a defective atToc132/120 receptor on the accumulation of chloroplast lipids. The mutant accumulated decreased amounts of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG). In the cold-acclimated mutant, PG accumulated at the control levels. However, 34:4-PG (18:3/16:1Δ3trans) was significantly decreased, which indicates that the mutant was impaired in synthesis of the chloroplast-derived PG. Major molecular species of MGDG and DGDG were significantly decreased, which was indicative of the decreased levels of triunsaturated fatty acids in galactolipids. The cold-acclimated mutant accumulated increased levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), which indicate that defect in the atToc132/120 receptor did not impair the ER pathway of lipid synthesis. Both cold-acclimated wildtype and mutant plants accumulated increased levels of phosphatidic acid (PA). The increased levels of major molecular species of PA suggest that some pool of PA was derived from degradation of both the chloroplast and extra-chloroplast lipids. The cold-acclimated mutant had decreased double bond index (DBI) and increased acyl chain length (ACL), which was indicative of decreased membrane fluidity. However, a decrease in the ratio of MGDG to DGDG indicate that the mutant was capable of remodeling membrane lipids in response to low temperatures. We conclude that the defective Toc132/120 receptor resulted in decreased synthesis of chloroplast lipids and decreased membrane fluidity.

Biological evaluation and chemoproteomics reveal potential antibacterial targets of a cajaninstilbene-acid analogue

Methicillin-resistant Staphylococcus aureus and the formation of persistent nongrowing subpopulations (persisters) is a serious threat to human. Our previous studies have proved that two cajaninstilbene acid (CSA) analogues, compound 5b and 5j display remarkable antibacterial activities, especially overcoming drug resistance of methicillin-resistant Staphylococcus aureus (MRSA). Present study found that 5b and 5j are capable of eradicating MRSA persisters. However, their underlying antibacterial mechanism is still obscure. In this study, biological evaluation was performed by transmission electron micrograph, membrane permeability and membrane depolarization experiment to reveal the effects of drugs on bacteria. Further, affinity-based protein profiling and transcriptional profiling were performed to characterise the protein targets in bacterial. Biological evaluation suggested that 5b has an effect on bacterial membrane, affinity-based protein profiling identified that 5b targets membrane associated protein PgsA and verified by in vitro labelling profile. Transcriptional profiling indicated that 5b interferes in phosphatidylglycerol (PG) synthesis pathway. This study identified a novel antibacterial target PgsA and it might be a potential target to combat the resistant bacteria.

Lipidomic consequences of phospholipid synthesis defects in Escherichia coli revealed by HILIC-ion mobility-mass spectrometry.

Our understanding of phospholipid biosynthesis in Gram-positive and Gram-negative bacteria is derived from the prototypical Gram-negative organism Escherichia coli. The inner and outer membranes of E. coli are largely composed of phosphatidylethanolamine (PE), minor amounts of phosphatidylglycerol (PG) and cardiolipin (CL). We report here the utility of hydrophilic interaction liquid chromatography (HILIC) paired with ion mobility-mass spectrometry (IM-MS) for the comprehensive analysis of the E. coli lipidome. Using strains with chromosomal deletions in the PG and CL synthesis genes pgsA and clsABC, respectively, we show that defective phospholipid biosynthesis in E. coli results in fatty-acid specific changes in select lipid classes and the presence of the minor triacylated phospholipids, acylphosphatidyl glycerol (acylPG) and N-acylphosphatidylethanolamine (N-acylPE). Notably, acylPGs were accumulated in the clsABC-KO strain, but were absent in other mutant strains. The separation of 1-lyso and 2-lyso-phosphatidylethanolamines (lysoPEs) is demonstrated in both the HILIC and IM dimensions. Using our previously validated calibration method, collision cross section values of nearly 200 phospholipids found in E. coli were determined on a traveling wave IM-MS platform, including newly reported values for cardiolipins, positional isomers of lysoPEs, acylPGs and N-acylPEs.

The PhoP/PhoQ two‐component regulatory system influences on Escherichia coli headgroup‐acylated glycerophospholipids

Headgroup‐acylated glycerophospholipids (GPLs) are a class of low abundance membrane lipids that are thought to play a roll in cell division and membrane integrity in Gram‐negative bacteria, such as Escherichia coli. The PhoP/PhoQ two‐component regulatory system is known to aid in virulence of pathogenic E. coli and responds to low Mg2+ concentrations. Previous research has suggested a protein activated by the PhoP/PhoQ system increases synthesis of acyl phosphatidylglycerol (acyl PG), a type of headgroup‐acylated GPL. Currently, though, the synthase of another headgroup‐acylated GPL, N‐acyl phosphatidylethanolamines (N‐acyl PE), is unknown. In order to determine if the PhoP/PhoQ system affects levels of acyl PGs, as well as N‐acyl PEs, wild‐type MG1655 E. coli with an empty pBAD 33.1 plasmid were grown in N‐minimal media with Mg2+ concentrations of either 8μM or 10mM. To confirm the activation of the PhoP/PhoQ system when cells are grown in 8μM Mg2+ media a reporter strain of E. coli, pNL3, was used. A β‐galactosidase assay of the pNL3 stain confirmed the activation of the PhoP/PhoQ system in low Mg2+ conditions through a 3.8 fold increase of activity in 8μM Mg2+ conditions compared to growths in 10mM Mg2+. In the MG1655 wild‐type strain, three normalized N‐acyl PE and four normalized acyl PG species were measured, utilizing reverse phase column mass spectrometry, and summed in each Mg2+ condition. N‐acyl PE and acyl PG levels in the 8μM Mg2+ growth was double that of the MG1655 grown in 10mM Mg2+. These data suggest that the PhoP/PhoQ system may be implicated in the synthesis of N‐acyl PEs and acyl PGs. Further research is required to verify the correlation between the activated PhoP/PhoQ system in N‐acyl PE and acyl PG synthase, as well as identify the protein specifically responsible for the synthesis of N‐acyl PEs.Support or Funding InformationSupported by National Science Foundation Major Research Instrumentation grant #1039659 and Research at Undergraduate Institution grant #1516805.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of novel lipid modifications and intermembrane dynamics in Corynebacterium glutamicum using high-resolution mass spectrometry [S

The complex cell envelopes of Corynebacterineae contribute to the virulence of pathogenic species (such as Mycobacterium tuberculosis and Corynebacterium diphtheriae) and capacity of nonpathogenic species (such as Corynebacterium glutamicum) to grow in diverse niches. The Corynebacterineae cell envelope comprises an asymmetric outer membrane that overlays the arabinogalactan-peptidoglycan complex and the inner cell membrane. Dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we have undertaken the first high-resolution analysis of C. glutamicum inner and outer membrane lipids. We identified 28 lipid (sub)classes (>233 molecular species), including new subclasses of acylated/acetylated trehalose mono/dicorynomycolic acids, using high-resolution LC/MS/MS coupled with mass spectral library searches in MS-DIAL. All lipid subclasses exhibited polarized distributions across the inner and outer membrane fractions generated by differential solvent extraction. Strikingly, deletion of the TmaT protein, which is required for transport of trehalose corynomycolates across the inner membrane, led to the accumulation of triacylglycerols in the inner membrane and to suppressed synthesis of phosphatidylglycerol and alanylated lipids. These analyses indicate unanticipated connectivity in the synthesis and/or transport of different lipid classes in C. glutamicum.

Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase 1

CDP diacylglycerol synthase (CDS) catalyses the conversion of phosphatidic acid (PA) to CDP-diacylglycerol, an essential intermediate in the synthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol (PI). CDS activity has been identified in mitochondria and endoplasmic reticulum of mammalian cells apparently encoded by two highly-related genes, CDS1 and CDS2. Cardiolipin is exclusively synthesised in mitochondria and recent studies in cardiomyocytes suggest that the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α and β) serve as transcriptional regulators of mitochondrial biogenesis and up-regulate the transcription of the CDS1 gene. Here we have examined whether CDS1 is responsible for the mitochondrial CDS activity. We report that differentiation of H9c2 cells with retinoic acid towards cardiomyocytes is accompanied by increased expression of mitochondrial proteins, oxygen consumption, and expression of the PA/PI binding protein, PITPNC1, and CDS1 immunoreactivity. Both CDS1 immunoreactivity and CDS activity were found in mitochondria of H9c2 cells as well as in rat heart, liver and brain mitochondria. However, the CDS1 immunoreactivity was traced to a peripheral p55 cross-reactive mitochondrial protein and the mitochondrial CDS activity was due to a peripheral mitochondrial protein, TAMM41, not an integral membrane protein as expected for CDS1. TAMM41 is the mammalian equivalent of the recently identified yeast protein, Tam41. Knockdown of TAMM41 resulted in decreased mitochondrial CDS activity, decreased cardiolipin levels and a decrease in oxygen consumption. We conclude that the CDS activity present in mitochondria is mainly due to TAMM41, which is required for normal mitochondrial function.

The mycobacterial Rv1551 glycerol-3-phosphate acyltransferase enhances phospholipid biosynthesis in cell lysates of Escherichia coli

Latent tuberculosis is caused by dormant Mycobacterium tuberculosis (Mtb) that is phenotypically tolerant to antibiotics. Dormant Mtb accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. The Rv1551 (PlsB1) gene is annotated as a putative glycerol-3-phosphate acyltransferase (GPAT) in the Mtb genome. GPAT catalyzes the first step of the glycerophospholipid biosynthetic pathway that synthesizes the lipid precursors for triacylglycerol biosynthesis. Although triacylglycerol biosynthesis is associated with Mtb dormancy, the functionality of the Rv1551 acyltransferase has not been investigated. We cloned the open reading frame of the Rv1551 acyltransferase and expressed it in Escherichia coli to study its function. We observed that E. coli cell lysates expressing Rv1551 displayed increased synthesis of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin from radiolabeled glycerol-3-phosphate and fatty acyl-coenzyme A precursors. When cultured in medium supplemented with long-chain fatty acids, E. coli expressing Rv1551 exhibited significantly higher viable cell counts during the exponential and stationary phases. These results suggest that Rv1551 displays function as a GPAT by enhancing the synthesis of phospholipids from exogenously provided fatty acids in E. coli cell lysates. This is the first report showing that Rv1551 is a functional GPAT that catalyzes the initial step of glycerophospholipid biosynthesis in the mycobacterial cell.

Overexpression of Glycerol-3-Phosphate Acyltransferase from Suaeda salsa Improves Salt Tolerance in Arabidopsis.

Glycerol-3-phosphate acyltransferase is the first acyl esterifying enzyme in phosphatidylglycerol (PG) synthesis process. In this study, we isolated and characterized the glycerol-3-phosphate acyltransferase (GPAT) gene from Suaeda salsa (S. salsa) and obtained the full length of the GPAT gene from S. salsa (SsGPAT) by 5′ and 3′ RACE. The clone contained an open reading frame (ORF) of 1167 bp nucleotides that comprised of 388 amino acid residues. Real-time PCR revealed that the mRNA accumulation of GPAT in S. salsa was induced by salt stress. The highest expression levels were observed when S. salsa leaves were exposed to 300 mM NaCl treatment. At the germination stage, the germination rate and root length of overexpressed Arabidopsis strains were significantly higher than WT under different concentrations of NaCl treatments, while the inhibitory effect was significantly severe in T-DNA insertion mutant strains. In the seedling stage, chlorophyll content, the photochemical efficiency of PSII, PSI oxidoreductive activity (ΔI/Io), and the unsaturated fatty acid content of PG decreased less in overexpressed strains and more in mutant strains than that in WT under salt stress. These results suggest that the overexpression of SsGPAT in Arabidopsis enhances salt tolerance and alleviates the photoinhibition of PSII and PSI under salt stress by improving the unsaturated fatty acid content of PG.

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana.

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.

Total synthesis and mass spectrometric analysis of a Mycobacterium tuberculosis phosphatidylglycerol featuring a two-step synthesis of (R)-tuberculostearic acid.

We report the total synthesis of (R)-tuberculostearic acid-containing Mycobacterium tuberculosis phosphatidylglycerol (PG). The approach features a two-step synthesis of (R)-tuberculostearic acid, involving an (S)-citronellyl bromide linchpin, and the phosphoramidite-assisted assembly of the full PG structure. Collision-induced dissociation mass spectrometry of two chemically-synthesized PG acyl regioisomers revealed diagnostic product ions formed by preferential loss of carboxylate at the secondary (sn-2) position.

In Vivo and in Vitro Synthesis of Phosphatidylglycerol by an Escherichia coli Cardiolipin Synthase

Phosphatidylglycerol (PG) makes up 5-20% of the phospholipids of Escherichia coli and is essential for growth in wild-type cells. PG is synthesized from the dephosphorylation of its immediate precursor, phosphatidylglycerol phosphate (PGP) whose synthase in E. coli is PgsA. Using genetic, biochemical, and highly sensitive mass spectrometric approaches, we identified an alternative mechanism for PG synthesis in E. coli that is PgsA independent. The reaction of synthesis involves the conversion of phosphatidylethanolamine and glycerol into PG and is catalyzed by ClsB, a phospholipase D-type cardiolipin synthase. This enzymatic reaction is demonstrated herein both in vivo and in vitro as well as by using the purified ClsB protein. When the growth medium was supplemented with glycerol, the expression of E. coli ClsB significantly increased PG and cardiolipin levels, with the growth deficiency of pgsA null strain also being complemented under such conditions. Identification of this alternative mechanism for PG synthesis not only expands our knowledge of bacterial anionic phospholipid biosynthesis, but also sheds light on the biochemical functions of the cls gene redundancy in E. coli and other bacteria. Finally, the PGP-independent PG synthesis in E. coli may also have important implications for the understanding of PG biosynthesis in eukaryotes that remains incomplete.

The action mechanism of daptomycin

Daptomycin is a lipopeptide antibiotic produced by the soil bacterium Streptomyces roseosporus that is clinically used to treat severe infections with Gram-positive bacteria. In this review, we discuss the mode of action of this important antibiotic. Although daptomycin is structurally related to amphomycin and similar lipopeptides that inhibit peptidoglycan biosynthesis, experimental studies have not produced clear evidence that daptomycin shares their action mechanism. Instead, the best characterized effect of daptomycin is the permeabilization and depolarization of the bacterial cell membrane. This activity, which can account for daptomycin’s bactericidal effect, correlates with the level of phosphatidylglycerol (PG) in the membrane. Accordingly, reduced synthesis of PG or its increased conversion to lysyl-PG promotes bacterial resistance to daptomycin. While other resistance mechanisms suggest that daptomycin may indeed directly interfere with cell wall synthesis or cell division, such effects still await direct experimental confirmation. Daptomycin’s complex structure and biosynthesis have hampered the analysis of its structure activity relationships. Novel methods of total synthesis, including a recent one that is carried out entirely on a solid phase, will enable a more thorough and systematic exploration of the sequence space.

Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants

Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement.

Effects of dehydration and rehydration on the leaf lipids and lipid metabolism in Parkinsonia aculeata (Caesalpiniaceae)

Plants of Parkinsonia aculeata L. were dehydrated by withholding water for 13 days and then rehydrated. The leaves of control plants and stressed plants were analyzed every four days from the beginning of stress and 6, 24, and 48 h after stress relief. Analysis of leaf water status showed a decrease in relative water content of nearly 16% during dehydration and a return to its control value after 48 h of rehydration. The contents of chlorophylls, chloroplast lipids (monogalactosyldiacylglycerol and digalactosyldiacylglycerol), and neutral lipids increased by 38%, 20%, and 125% (in μg·mg dry mass−1), respectively, when the daily decrease of relative water content reached 1.4%, while the mass of extra-plastidial lipids (phosphatidylcholine and phosphatidylethanolamine) increased by 25% (in μg·mg dry mass−1) only when the daily water loss was 2.6%. In addition, incorporation of radioactive acetate in lipids showed a preferential synthesis of digalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The unsaturation degree of galactolipids remained stable, while a slight increase of unsaturation of phosphatidylcholine and phosphatidylethanolamine was observed. After rehydration, the mass of chlorophylls fell sharply, while the amount of total lipids decreased slowly as the level remained 17% higher than in the control 6 h after stress relief. Our results show that Parkinsonia plants are sensitive to a slight variation of the water potential and respond with an increased synthesis of membrane lipids during stress and after rehydration.

Synthesis and Characterization of Picket Porphyrin Receptors That Bind Phosphatidylglycerol, an Anionic Phospholipid Found in Bacterial Membranes

The lipid binding ability of four urea-picket porphyrins designed to bind to both the phosphate anion portion as well as the glycerol hydroxyl groups of phosphatidylglycerol (PG) has been investigated. Isothermal titration calorimetry (ITC) and (1)H NMR were used to determine the receptor's stoichiometry of binding, association constants, and both the enthalpy and entropy of binding with the PG anion. Spectral evidence shows that the phosphate anion portion of PG is hydrogen bonded to the urea groups of the receptors. This binding interaction orients the PG anion in the receptor pocket such that its glycerol hydroxyl groups can align with a third urea picket, and results are furnished that suggest this multifunctional interaction does occur. The structure of the entire picket was found to influence the enthalpy and entropy of lipid binding. The synthesis of tetrabutlyammonium phosphatidylglycerol (TBAPG), and a detailed spectral characterization of its headgroup, is also presented.

Palmitoleate Is a Mitogen, Formed upon Stimulation with Growth Factors, and Converted to Palmitoleoyl-phosphatidylinositol

Controversial correlations between biological activity and concentration of the novel lipokine palmitoleate (9Z-hexadecenoate, 16:1) might depend on the formation of an active 16:1 metabolite. For its identification, we analyzed the glycerophospholipid composition of mouse Swiss 3T3 fibroblasts in response to 16:1 using LC-MS/MS. 16:1 was either supplemented to the cell culture medium or endogenously formed when cells were stimulated with insulin or growth factors as suggested by the enhanced mRNA expression of 16:1-biosynthetic enzymes. The proportion of 1-acyl-2-16:1-sn-phosphatidylinositol (16:1-PI) was time-dependently and specifically increased relative to other glycerophospholipids under both conditions and correlated with the proliferation of fatty acid (16:1, palmitate, oleate, or arachidonate)-supplemented cells. Accordingly, cell proliferation was impaired by blocking 16:1 biosynthesis using the selective stearoyl-CoA desaturase-1 inhibitor CAY10566 and restored by supplementation of 16:1. The accumulation of 16:1-PI occurred throughout cellular compartments and within diverse mouse cell lines (Swiss 3T3, NIH-3T3, and 3T3-L1 cells). To elucidate further whether 16:1-PI is formed through the de novo or remodeling pathway of PI biosynthesis, phosphatidate levels and lyso-PI-acyltransferase activities were analyzed as respective markers. The proportion of 16:1-phosphatidate was significantly increased by insulin and growth factors, whereas lyso-PI-acyltransferases showed negligible activity for 16:1-coenzyme A. The relevance of the de novo pathway for 16:1-PI biosynthesis is supported further by the comparable incorporation rate of deuterium-labeled 16:1 and tritium-labeled inositol into PI for growth factor-stimulated cells. In conclusion, we identified 16:1 or 16:1-PI as mitogen whose biosynthesis is induced by growth factors.

Simple Thermodynamically-Derived Model for Predicting the Hydrolase and Transferase Activity of Phospholipase D in the Synthesis of Phosphatidylglycerol

Preparation of a single and pure phospholipid via transphosphatidylation has been a much sought after endeavor in the pharmaceutical and nonmedical industries. For this reason, phosphatidylglycerol, a lung surfactant, was produced from phosphatidylcholine with defined fatty acids, ie, dipalmitoyl phosphatidylcholine. Substrate type and concentration, enzyme source, and reaction temperature were investigated. Phospholipase D from two sources, ie, savoy cabbage, was purified in the authors’ laboratory and a commercially available Streptomyces species was used for this study. The substrates used were glycerol, a polyhydric alcohol, and solketal, a monohydric form of glycerol. The progress of the reaction was monitored using thin layer chromatography, and synthesis with solketal, an unusual form of glycerol, was confirmed by liquid chromatography mass spectrometry. Surface response methodology used on four combinations of enzyme and substrate at various temperatures (30 °C–60 °C) and concentration (0.25–1 mM) revealed that yield and selectivity was temperature-driven and predictable. To validate further the thermodynamic attributes, a modified version of the Eyring equation was derived from selectivity and the Arhenius equation. These equations provide some useful insights into the difference in activation of enthalpy change(ΔΔH++) and difference in activation of entropy change(ΔΔS++). Plots of ln[PG]/[PA] versus 1/T gave good linear fits for these four combinations. In addition, a new thermodynamic parameter known as TPG = PAhas emerged as a theoretical temperature for equivalent transferase and hydrolase activity.