Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).     

Towards the genetic engineering of triacylglycerols of defined fatty acid composition: major changes in erucic acid content at the sn-2 position affected by the introduction of a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii into oil seed rape

A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.     

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.     

Substrate specificities of the membrane-bound and partially purified microsomal acyl-CoA:1-acylglycerol-3-phosphate acyltransferase from etiolated shoots of Pisum sativum (L.)

Membrane fractions enriched in rough endoplasmic reticulum and not contaminated with plastidial membranes were isolated from etiolated shoots of Pisum sativum (L.). From these fractions the acyl-CoA:1-acyl-sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.51) was solubilized by extracting the membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate at high ionic strength. The subsequent separation of the solubilized fractions on a Mono Q column resulted in a tenfold enriched enzymic activity, which could be stabilized by polyethyleneglycol precipitation. A comparison of the substrate specificities and selectivities of the solubilized, enriched 1-acylglycerol-3-phosphate acyltransferase and the corresponding membrane-bound activity revealed no appreciable difference. Both enzymic forms specifically utilized acyl-CoA thioesters as acyl donors whereas the corresponding acyl-acyl carrier protein thioesters were not used. Furthermore, the membrane-bound as well as the solubilized enriched form showed not only higher activities with 1-oleoylthan with 1-palmitoylglycerol-3-phosphate but also pronounced specificities and selectivities for unsaturated C₁₈-CoA thioesters. Hence, the extraplastidial 1-acylglycerol-3-phosphate acyltransferase which catalyses the formation of phosphatidic acid with an eukaryotic fatty-acid pattern was partially purified.Abbreviations ACP acyl carrier protein - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate - LPA-AT acyl-CoA:1-acylglycerol-3-phosphate acyltransferase - PEG polyethyleneglycol The authors are grateful to the Deutsche Forschungsgemeinschaft for financial support. We wish to thank Miss Ute Hammer for the analysis of the lipid composition of the microsomal fractions.     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).     

Identification of sn-Glycerol-1-phosphate Dehydrogenase Activity from Genomic Information on a Hyperthermophilic Archaeon,Sulfolobus tokodaii Strain 7

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.     

Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes

The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing V_MAX and K_M for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1.     

Cell envelope proteins involved in the transport of maltose and sn-glycerol-3-phosphate in Escherichia coli

Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the K_m of the transport system without influencing the V_max of substrate flux. The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective in sn-glycerol-3-phosphate transport.     

Purification and cDNA sequencing of an oleate-selective acyl-ACP:sn-glycerol-3-phosphate acyltransferase from pea chloroplasts

The soluble acyl-ACP:sn-glycerol-3-phosphate acyltransferase from chloroplasts of chilling-sensitive and -resistant plants differ in their fatty acid selectivity. Enzymes from resistant plants discriminate against non-fluid palmitic acid and select oleic acid whereas the acyltransferase from sensitive plants accepts both fatty acids. To use this difference for improving plant chilling resistance by biotechnology the gene for an oleate-selective enzyme is required. Therefore, the oleate-selective enzyme from pea seedlings was purified to apparent homogeneity. Tryptic peptides of internal origin were sequenced. Polyclonal antibodies raised in rabbits were used for an immunological screening of a pea leaf cDNA expression library in gt11. A positive clone of 1800 bp was selected showing an open reading frame which codes for 457 amino acids. The deduced amino acid sequence coincides perfectly with the tryptic sequences. A tentative assignment of the processing site was made which divides the preprotein into a mature protein of 41 kDa in accordance with experimental findings and a transit peptide of 88 amino acids. At present the comparison between a selective (pea) and an unselective (squash) acyltransferase sequence does not provide a clue for recognizing the structural differences resulting in different selectivities.     

SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Delta cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Delta and slc4Delta cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Delta and slc4Delta cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Delta and slc4Delta cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.     

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate,a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-²H₂]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2–3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Novel stereoconfiguration in lyso-bis-phosphatidic acid of cultured BHK-cells

Lyso-bis-phosphatidic acid purified from cultured hamster kidney fibroblast cells (BHK-cells) was subjected to strong alkaline hydrolysis. The hydrolysate contained phosphorus, free glycerol, total glycerol, α-glycerophosphate, β-glycerophosphate and sn-glycerol-3-phosphate in mole ratios of 1.0:1.0:1.9:0.4:0.6:0.02. The absence of sn-glycerol-3-phosphate indicates that the backbone of this lipid has the uncommon structure of 1-sn-glycerophosphoryl-1′-sn-glycerol. Consequently, the biosynthesis and the degradation of this lipid must differ from the other known mammalian glycerophospholipids.     

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.     

A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity - inside liposomes - of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within — which corresponds to the autopoietic growth mechanism.     

Formation of phospholipids from sn-glycerol-3-phosphate and free fatty acids or their derivatives by homogenates of soybean cotyledons

Cell-free homogenates of soybean cotyledons contain a sn-glycerol-3-phosphate acyltransferase system which incorporated [U-¹⁴C]-sn-glycerol-3-phosphate into 5 labelled lipids when incubated with palmitic acid in the presence of ATP and CoA. In decreasing order of incorporation of label, the lipids were: lysophosphatidic acid, monoacylglycerol, phosphatidic acid, diacylglycerol and triacylglycerol. The substrate specificity of the acyltransferase system was investigated with the fatty acids shown in order of decreasing rates of reaction; palmitate > stearate > oleate > linoleate > linolenate > laurate. Making these acids more soluble as triethanolamine salts or as polyoxyethylene sorbitan esters did not greatly enhance these rates of reaction. Activity was found in a 10000 g pellet containing plastids, mitochondria and glyoxysomes and also in the lipid layer; the activity in these particulate fractions was enhanced by the addition of cytosol which itself had little activity when gentle methods of cell disruption were used. During cotyledon development the total acyltransferase activity increased, although its specific activity slowly declined due to more rapid synthesis of other proteins. During germination total activity decreased but there was a transient increase in specific activity due to more rapid degradation of other proteins.     

Membrane glycerophospholipid biosynthesis in Neisseria meningitidis and Neisseria gonorrhoeae: identification, characterization, and mutagenesis of a lysophosphatidic acid acyltransferase

Lysophosphatidic acid (LPA) acyltransferases of Neisseria meningitidis and Neisseria gonorrhoeae were identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. In Escherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1-acyl-sn-glycerol-3-phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916-generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916 insertion in the meningococcal mutant was localized within a 771 bp open reading frame. The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. In N. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designated nIaA (n eisserial L PA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity,>40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcal nIaA gene complemented in trans a temperature-sensitive E. coli PlsC^ts? mutant. Tn916 and Ω-cassette insertional inactivations of the neisserial nIaA genes altered the membrane glycerophospholipid compositions of both N. meningitidis and N. gonorrhoeae but were not lethal. Therefore, the pathogenic Neisseria spp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcal nIaA mutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcal nIaA mutant, was increased in the meningococcal nIaA mutant. We postulate that the pathogenic Neisseria spp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence of nIaA enzymatic activity.Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on capsule and piliation.