Background: Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine.
Results: Plant LPCATs were expressed in yeast and biochemically characterized.
Conclusion: LPCATs can edit acyl composition of phosphatidylcholine through their combined forward and reverse reactions.
Significance: Plant LPCATs play a role in editing both sn-positions of PC and remove ricinoleic acid with high selectivity from this lipid.
Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.
Arabidopsis; Enzymes; Lipid Metabolism; Phosphatidylcholine; Plant Biochemistry; Lysophosphatidylcholine Acyltransferases; Phosphatidylcholine Metabolism; Plant Lipid Biochemistry; Enzyme
Cellular membranes contain glycerophospholipids, which have important structural and functional roles in cells. Glycerophospholipids are first formed in the de novo pathway (Kennedy pathway) and are matured in the remodeling pathway (Lands’ cycle). Recently, lysophospholipid acyltransferases functioning in Lands’ cycle were identified and characterized. Several enzymes involved in glycerophospholipid biosynthesis have been reported to have important roles in adipocytes. However, the role of Lands’ cycle in adipogenesis has not yet been reported. Using C3H10T1/2, a cell line capable of differentiating to adipocyte-like cells in vitro, changes of lysophospholipid acyltransferase activities were investigated. Lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylserine acyltransferase (LPSAT) activities were enhanced, especially with 18:2-CoA and 20:4-CoA as donors. Correspondingly, mRNA expression of LPCAT3, which possesses LPCAT, LPEAT and LPSAT activities with high specificity for 18:2- and 20:4-CoA, was upregulated during adipogenesis. Analysis of acyl-chain compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) showed a change in their profiles between preadipocytes and adipocytes, including an increase in the percentage of arachidonic acid-containing phospholipids. These changes are consistent with the activities of LPCAT3. Therefore, it is possible that enhanced phospholipid remodeling by LPCAT3 may be associated with adipocyte differentiation.
glycerophospholipid; lysophospholipid acyltransferase; adipocyte; C3H10T1/2; LPCAT3; Lands’ cycle; arachidonic acid
The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p<10−8) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [14C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p<10−5) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.
Colorectal cancer; Lysophosphatidic acyltransferase; Microarrays; Lipid metabolism; Phosphatidylcholine
Respiratory distress syndrome (RDS), which is the leading cause of death in premature infants, is caused by surfactant deficiency. The most critical and abundant phospholipid in pulmonary surfactant is saturated phosphatidylcholine (SatPC), which is synthesized in alveolar type II cells de novo or by the deacylation-reacylation of existing phosphatidylcholine species. We recently cloned and partially characterized a mouse enzyme with characteristics of a lung lysophosphatidylcholine acyltransferase (LPCAT1) that we predicted would be involved in surfactant synthesis. Here, we describe our studies investigating whether LPCAT1 is required for pulmonary surfactant homeostasis. To address this issue, we generated mice bearing a hypomorphic allele of Lpcat1 (referred to herein as Lpcat1GT/GT mice) ufsing a genetrap strategy. Newborn Lpcat1GT/GT mice showed varying perinatal mortality from respiratory failure, with affected animals demonstrating hallmarks of respiratory distress such as atelectasis and hyaline membranes. Lpcat1 mRNA levels were reduced in newborn Lpcat1GT/GT mice and directly correlated with SatPC content, LPCAT1 activity, and survival. Surfactant isolated from dead Lpcat1GT/GT mice failed to reduce minimum surface tension to wild-type levels. Collectively, these data demonstrate that full LPCAT1 activity is required to achieve the levels of SatPC essential for the transition to air breathing.
Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen that multiplies within a parasitophorous vacuole called an inclusion. We report that the location of several host-cell proteins present in the cytosol, the nucleus, and membranes was altered during Ct development. The acyl-CoA synthetase enzyme ACSL3 and the soluble acyl-CoA binding protein ACBD6 were mobilized from organelle membranes and the nucleus, respectively, into the lumen of the inclusion. The nuclear protein ZNF23, a pro-apoptosis factor, was also translocated into the inclusion lumen. ZNF23, among other proteins, might be targeted by Ct to inhibit host cell apoptosis, thereby enabling bacterial survival. In contrast, the acyl-CoA:lysophosphatidylcholine acyltransferase LPCAT1, an endoplasmic reticulum membrane protein, was recruited to the inclusion membrane. The coordinated action of ACBD6, ACSL3 and LPCAT1 likely supports remodeling and scavenging of host lipids into bacterial-specific moieties essential to Ct growth. To our knowledge, these are the first identified host proteins known to be intercepted and translocated into the inclusion.
Escherichia coli apolipoprotein N-acyltransferase (Lnt) transfers an acyl group from sn-1-glycerolphospholipid to the free α-amino group of the N-terminal cysteine of apolipoproteins, resulting in mature triacylated lipoprotein. Here we report that the Lnt reaction proceeds through an acyl enzyme intermediate in which a palmitoyl group forms a thioester bond with the thiol of active site residue C387 that was cleaved by neutral hydroxylamine. Lnt(C387S) also formed a fatty acyl intermediate that was resistant to neutral hydroxylamine treatment, consistent with formation of an oxygen-ester linkage. Lnt(C387A) did not form an acyl enzyme intermediate and, like Lnt(C387S), did not have any detectable Lnt activity, indicating that acylation can not occur at other positions in the catalytic domain. The existence of this thioacyl-enzyme intermediate allowed us to determine whether essential residues in the catalytic domain of Lnt affect the first step of the reaction, the formation of the acyl enzyme intermediate, or the second step in which the acyl chain is transferred to apolipoprotein substrate. In the catalytic triad, E267 is required for the formation of the acyl-enzyme intermediate, indicating its role in enhancing the nucleophilicity of C387. E343 is also involved in the first step but is not in close proximity to the active site. W237, Y388 and E389 play a role in the second step of the reaction since acyl-Lnt is formed but N-acylation does not occur. The data presented allow discrimination between the functions of essential Lnt residues in catalytic activity and substrate recognition.
Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.
Arabidopsis; diacylglycerol acyltransferase; fatty acid; oilseed; oleic acid; Thraustochytrium aureum; triacylglycerol
The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution.
The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.
mouse long-chain acyl-CoA thioesterase Acot7; 4HBT domain
Recent studies have suggested that formation of Golgi membrane tubules
involves the generation of membrane-associated lysophospholipids by a
cytoplasmic Ca2+-independent phospholipase A2
(PLA2). Herein, we provide additional support for this idea by
showing that inhibition of lysophospholipid reacylation by a novel
Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the
rapid tubulation of Golgi membranes, leading in their retrograde movement to
the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by
2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously
characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect
of CI-976 was similar to that of brefeldin A, except that the coatomer subunit
β-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced
the cytosol-dependent formation of tubules from Golgi complexes in vitro and
increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover,
preincubation of cells with PLA2 antagonists inhibited the ability
of CI-976 to induce tubules. These results suggest that Golgi membrane tubule
formation can result from increasing the content of lysophospholipids in
membranes, either by stimulation of a PLA2 or by inhibition of an
LPAT. These two opposing enzyme activities may help to coordinately regulate
Golgi membrane shape and tubule formation.
Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. We used expression cloning to identify cDNAs encoding a wax synthase in the mouse preputial gland. The wax synthase gene is located on the X chromosome and encodes a member of the acyltransferase family of enzymes that synthesize neutral lipids. Expression of wax synthase in cultured cells led to the formation of wax monoesters from straight chain saturated, unsaturated, and polyunsaturated fatty alcohols and acids. Polyisoprenols also were incorporated into wax monoesters by the enzyme. The wax synthase had little or no ability to synthesize cholesteryl esters, diacylglycerols, or triacylglycerols, whereas other acyltransferases, including the acyl-CoA:monoacylglycerol acyltransferase 1 and 2 enzymes and the acyl-CoA:diacylglycerol acyltransferase 1 and 2 enzymes, exhibited modest wax monoester synthesis activities. Confocal light microscopy indicated that the wax synthase was localized in membranes of the endoplasmic reticulum. Wax synthase mRNA was abundant in tissues rich in sebaceous glands such as the preputial gland and eyelid and was present at lower levels in other tissues. Coexpression of cDNAs specifying fatty acyl-CoA reductase 1 and wax synthase led to the synthesis of wax monoesters. The data suggest that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.
Enzymes of the membrane-bound O-acyltransferase (MBOAT) family add fatty acyl chains to a diverse range of protein and lipid substrates. A chromosomal translocation disrupting human MBOAT1 results in a novel syndrome characterized by male sterility and brachydactyly. We have found that the Drosophila homologues of MBOAT1, Oysgedart (Oys), Nessy (Nes), and Farjavit (Frj), are lysophospholipid acyltransferases. When expressed in yeast, these MBOATs esterify specific lysophospholipids preferentially with unsaturated fatty acids. Generating null mutations for each gene allowed us to identify redundant functions for Oys and Nes in two distinct aspects of Drosophila germ cell development. Embryos lacking both oys and nes show defects in the ability of germ cells to migrate into the mesoderm, a process guided by lipid signals. In addition, oys nes double mutant adult males are sterile due to specific defects in spermatid individualization. oys nes mutant testes, as well as single, double, and triple mutant whole adult animals, show an increase in the saturated fatty acid content of several phospholipid species. Our findings suggest that lysophospholipid acyltransferase activity is essential for germline development and could provide a mechanistic explanation for the etiology of the human MBOAT1 mutation.
DesT is a transcriptional repressor that regulates the genes which control the unsaturated:saturated fatty acid ratio available for membrane lipid synthesis. DesT bound to unsaturated acyl-CoA has a high affinity for its cognate palindromic DNA-binding site, whereas DesT bound to saturated acyl-CoA does not bind to this site. Structural analyses of the DesT–oleoyl-CoA–DNA and DesT–palmitoyl-CoA complexes reveal that acyl chain shape directly influences the packing of hydrophobic core residues within the DesT ligand-binding domain. These changes are propagated to the paired DNA-binding domains via conformational changes to modulate DNA binding. These structural interpretations are supported by the in vitro and in vivo characterization of site-directed mutants. The regulation of DesT by the unsaturated:saturated ratio of acyl chains rather than the concentration of a single ligand is a paradigm for understanding transcriptional regulation of membrane lipid homeostasis.
A phospholipase A2 was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A2 (LPLA2) and subsequently designated group XV phospholipase A2. LPLA2 has 49 percent of amino acid sequence identity to lecithin cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA2 is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA2−/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA2−/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA2 may play a primary role in phospholipid homeostasis, drug toxicity, and host defense.
Lysosomal phospholipase A2 (LPLA2); 1-O-acylceramide synthase; phospholipase A2; transacylase; amiodarone; cationic amphiphilic drugs; lecithin-cholesterol acyltransferase (LCAT); LCAT like lysophospholipase (LLPL); alveolar macrophages (AMs); pulmonary surfactant; phospholipidosis
Members of the adenylate-forming family of enzymes play a role in the metabolism of halogenated aromatics and of short, medium, and long chain fatty acids, as well as in the biosynthesis of menaquinone, peptide antibiotics, and peptide siderophores. This family includes a subfamily of acyl- and aryl-CoA ligases that catalyze thioester synthesis through two half-reactions. A carboxylate substrate first reacts with ATP to form an acyl-adenylate. Subsequent to the release of the product PPi, the enzyme binds CoA, which attacks the activated acyl group to displace AMP. Structural and functional studies on different family members suggest that these enzymes alternate between two conformations during catalysis of the two half-reactions. Specifically, after the initial adenylation step, the C-terminal domain rotates by ~140° to adopt a second conformation for thioester formation. Previously, we determined the structure of 4-chlorobenzoate:CoA ligase (CBL) in the adenylate forming conformation bound to 4-chlorobenzoate. We have determined two new crystal structures. We have determined the structure of CBL in the original adenylate-forming conformation, bound to the adenylate intermediate. Additionally, we have used a novel product analog, 4-chlorophenacyl-CoA, to trap the enzyme in the thioester-forming conformation and determined this structure in a new crystal form. This work identifies a novel binding pocket for the CoA nucleotide. The structures presented herein provide the foundation for biochemical analyses presented in the accompanying manuscript (Wu et al.). The complete characterization of this enzyme allows us to provide an explanation for the use of the domain alternation strategy by these enzymes.
4-chorobenzoate: CoA ligase; 4-chlorobenzoate; coenzyme A; adenylate-forming enzyme superfamily; acyl-adenylate; X-ray structure; Domain Alternation; enzyme conformational changes
In the yeast Saccharomyces cerevisiae triacylglycerols (TAG) are synthesized by the acyl-CoA dependent acyltransferases Dga1p, Are1p, Are2p and the acyl-CoA independent phospholipid:diacylglycerol acyltransferase (PDAT) Lro1p which uses phosphatidylethanolamine (PE) as a preferred acyl donor. In the present study we investigated a possible link between TAG and PE metabolism by analyzing the contribution of the four different PE biosynthetic pathways to TAG formation, namely de novo PE synthesis via Psd1p and Psd2p, the CDP-ethanolamine (CDP-Etn) pathway and lyso-PE acylation by Ale1p. In cells grown on the non-fermentable carbon source lactate supplemented with 5 mM ethanolamine (Etn) the CDP-Etn pathway contributed most to the cellular TAG level, whereas mutations in the other pathways displayed only minor effects. In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDP-Etn pathway both the cellular and the microsomal levels of PE were markedly decreased, whereas in other mutants of PE biosynthetic routes depletion of this aminoglycerophospholipid was less pronounced in microsomes. This observation is important because Lro1p similar to the enzymes of the CDP-Etn pathway is a component of the ER. We conclude from these results that in cki1∆dpl1∆eki1∆ insufficient supply of PE to the PDAT Lro1p was a major reason for the strongly reduced TAG level. Moreover, we found that Lro1p activity was markedly decreased in cki1∆dpl1∆eki1∆, although transcription of LRO1 was not affected. Our findings imply that (i) TAG and PE syntheses in the yeast are tightly linked; and (ii) TAG formation by the PDAT Lro1p strongly depends on PE synthesis through the CDP-Etn pathway. Moreover, it is very likely that local availability of PE in microsomes is crucial for TAG synthesis through the Lro1p reaction.
► CDP-Etn pathway provides PE for Lro1p. ► Depletion of PE in ER causes low TAG. ► Link between phospholipid and TAG metabolism.
CF, cellular fraction; CL, cardiolipin; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase; DMPE, dimethylphosphatidylethanolamine; ER, endoplasmic reticulum; Etn, ethanolamine; LP, lipid particle; LPL, lysophospholipid(s); MMGlu, minimal glucose media; MMLac, minimal lactate media; MAM, mitochondria associated membrane; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PDAT, phospholipid:diacylglycerol acyltransferase; RT-PCR, reverse transcription polymerase chain reaction; SE, steryl ester; TAG, triacylglycerol; TLC, thin-layer chromatography; YPD, complex glucose media; YPLac, complex lactate media; Phosphatidylethanolamine; Triacylglycerol; Acyltransferase; CDP-ethanolamine; Yeast; Saccharomyces cerevisiae
Two previously uncharacterized Arabidopsis genes that encode proteins with acyltransferase PlsC regions were selected for study based on their sequence similarity to a recently identified lung lysophosphatidylcholine acyltransferase (LPCAT). To identify their substrate specificity and biochemical properties, the two Arabidopsis acyltransferases, designated AtLPEAT1, (At1g80950), and AtLPEAT2 (At2g45670) were expressed in yeast knockout lines ale1 and slc1 that are deficient in microsomal lysophosphatidyl acyltransferase activities.
Expression of AtLPEAT1 in the yeast knockout ale1 background exhibited strong acylation activity of lysophosphatidylethanolamine (LPE) and lysophosphatidate (LPA) with lower activity on lysophosphatidylcholine (LPC) and lysophosphatidylserine (LPS). AtLPEAT2 had specificities in the order of LPE > LPC > LPS and had no or very low activity with LPA. Both acyltransferases preferred 18:1-LPE over 16:0-LPE as acceptor and preferred palmitoyl-CoA as acyl donor in combination with 18:1-LPE. Both acyltransferases showed no or minor responses to Ca2+, despite the presence of a calcium binding EF-hand region in AtLPEAT2. AtLPEAT1 was more active at basic pH while AtLPEAT2 was equally active between pH 6.0 – 9.0.
This study represents the first description of plant acyltransferases with a preference for LPE. In conclusion it is suggested that the two AtLPEATs, with their different biochemical and expression properties, have different roles in membrane metabolism/homoeostasis.
We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neoformans var. neoformans, under native conditions, by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography (TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of the reaction between substrate phosphatidylcholine (PC) and cryptococcal culture supernatants indicating the presence of phospholipase B (PLB). No lysophosphatidylcholine (lyso-PC) or products indicative of phospholipase C, phospholipase D, or other lipase activity were identified. Use of PC and lyso-PC containing radiolabelled acyl chains and separation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identified by the formation of radioactive PC from lyso-PC. Extracellular enzyme production was maximal after 6 to 10 h in fresh medium. Assay conditions were optimized for pH, linearity with time, enzyme concentration, and saturation by substrates to allow comparison with phospholipases from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (+/- standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 micromol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing substrate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyso-PC greater than 200 microM. Enzyme activities were stable at acid pH (3.8), with pH optima of 3.5 to 4.5. Activities were unchanged in the presence of exogenous serine protease inhibitors, divalent cations, and EDTA. We conclude that C. neoformans produces highly active extracellular PLB, LPL, and LPTA under native conditions.
The glycerol-3-phosphate acyltransferase Sct1p/Gat2p is shown to regulate fatty acyl chain desaturation by competing with the fatty acid desaturase Ole1p for C16:0-CoA. The activity of Sct1p depends on the level of expression and the phosphorylation state. The acyltransferase Cst26p regulates the phosphorylation of Sct1p.
The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Taking advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.
The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-013-1870-8) contains supplementary material, which is available to authorized users.
PDAT; DGAT; Sunflower; Safflower; Microsomal preparation; Triacylglycerols; Lipids
The mitochondrial dimeric phospholipid cardiolipin is characterized by a
high degree of unsaturation of its acyl chains, which is important for its
functional interaction with mitochondrial enzymes. The unusual fatty acid
composition of cardiolipin molecular species emerges from a de novo
synthesized “premature” species by extensive acyl chain remodeling
that involves as yet only partially identified acyltransferases and
phospholipases. Recently, the yeast protein Taz1p was shown to function as a
transacylase, which catalyzes the reacylation of monolysocardiolipin to mature
cardiolipin. A defect in the orthologous human TAZ gene is associated
with Barth syndrome, a severe genetic disorder, which may lead to cardiac
failure and death in childhood. We now identified the protein encoded by
reading frame YGR110W as a mitochondrial phospholipase, which
deacylates de novo synthesized cardiolipin. Ygr110wp has a strong
substrate preference for palmitic acid residues and functions upstream of
Taz1p, to generate monolysocardiolipin for Taz1p-dependent reacylation with
unsaturated fatty acids. We therefore rename the Ygr110wp as Cld1p
(cardiolipin-specific deacylase 1).
Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6.
Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms.
The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.
AIM: To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene transcription.
METHODS: Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation.
RESULTS: Lpcat1 translocates into the nucleus from the cytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli, two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overexpressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.
CONCLUSION: These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.
Lipopolysaccharide; Nuclear import; Lysophosphatidylcholine acyltransferase 1; Gene expression; Lung epithelia; Epigenetic code; Quantitative reverse transcription polymerase chain reaction; Haemophilus influenza; Escherichia coli
Lithocholic acid (LCA) is an endogenous compound associated with hepatic toxicity during cholestasis. LCA exposure in mice resulted in decreased serum lysophosphatidylcholine (LPC) and sphingomyelin levels due to elevated lysophosphatidylcholine acyltransferase (LPCAT) and sphingomyelin phosphodiesterase (SMPD) expression. Global metabolome analysis indicated significant decreases in serum palmitoyl-, stearoyl-, oleoyl- and linoleoyl-LPC levels after LCA exposure. LCA treatment also resulted in decreased serum sphingomyelin levels and increased hepatic ceramide levels, and induction of LPCAT and SMPD mRNAs. Transforming growth factor-β TGF-β) induced Lpcat2/4 and Smpd3 gene expression in primary hepatocytes and the induction was diminished by pretreatment with the SMAD3 inhibitor SIS3. Furthermore, alteration of the LPC metabolites and Lpcat1/2/4 and Smpd3 expression was attenuated in LCA-treated farnesoid X receptor-null mice that are resistant to LCA-induced intrahepatic cholestasis. This study revealed that LCA induced disruption of phospholipid/sphingolipid homeostasis through TGF-β signaling and that serum LPC is a biomarker for biliary injury.
Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.
Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.