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Andreas Conzelmann - One of the best experts on this subject based on the ideXlab platform.
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topology of 1 acyl sn glycerol 3 phosphate Acyltransferases slc1 and ale1 and related membrane bound o Acyltransferases mboats of saccharomyces cerevisiae
Journal of Biological Chemistry, 2011Co-Authors: Martin Pagac, Carole Roubaty, Hector Vazquez De La Mora, Cecile Duperrex, Christine Vionnet, Andreas ConzelmannAbstract:In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate Acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-Acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol Acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate Acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed.
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slc1 and slc4 encode partially redundant acyl coenzyme a 1 acylglycerol 3 phosphate o Acyltransferases of budding yeast
Journal of Biological Chemistry, 2007Co-Authors: Mohammed Benghezal, Carole Roubaty, Vijayanath Veepuri, Jens Knudsen, Andreas ConzelmannAbstract: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 slc1Δ cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Δ and slc4Δ cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Δ and slc4Δ 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 slc1Δ and slc4Δ 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.
Stephen L Sturley - One of the best experts on this subject based on the ideXlab platform.
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sterol and diacylglycerol Acyltransferase deficiency triggers fatty acid mediated cell death
Journal of Biological Chemistry, 2009Co-Authors: Jeanne Garbarino, Mahajabeen Padamsee, Lisa Wilcox, Diana Dambrosio, Nicole Ramsey, Omar J Jabado, Aaron Turkish, Peter Oelkers, Kelly V Ruggles, Stephen L SturleyAbstract:Deletion of the Acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol Acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast Acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic β-cell dysfunction, diabetes, and the metabolic syndrome.
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a lecithin cholesterol Acyltransferase like gene mediates diacylglycerol esterification in yeast
Journal of Biological Chemistry, 2000Co-Authors: Peter Oelkers, Arthur H Tinkelenberg, Naz Erdeniz, Debra Cromley, Jeffrey T Billheimer, Stephen L SturleyAbstract:Abstract The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian Acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol Acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol Acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in theLRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol Acyltransferase. These mechanisms may also exist in mammalian cells.
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expression in yeast and tobacco of plant cdnas encoding acyl coa diacylglycerol Acyltransferase
FEBS Journal, 2000Co-Authors: Pierrette Bouviernave, Stephen L Sturley, Peter Oelkers, Pierre Benveniste, Hubert SchallerAbstract:During the course of a search for cDNAs encoding plant sterol Acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol Acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol Acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol Acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol Acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2–3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol Acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol Acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol Acyltransferase-transformed plants.
Peter Oelkers - One of the best experts on this subject based on the ideXlab platform.
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four Acyltransferases uniquely contribute to phospholipid heterogeneity in saccharomyces cerevisiae
Lipid Insights, 2016Co-Authors: Peter Oelkers, Keshav PokhrelAbstract:Diverse acyl-CoA species and Acyltransferase isoenzymes are components of a complex system that synthesizes glycerophospholipids and triacylglycerols. Saccharomyces cerevisiae has four main acyl-CoA species, two main glycerol-3-phosphate 1-O-Acyltransferases (Gat1p, Gat2p), and two main 1-acylglycerol-3-phosphate O-Acyltransferases (Lpt1p, Slc1p). The in vivo contribution of these isoenzymes to phospholipid heterogeneity was determined using haploids with compound mutations: gat1Δlpt1Δ, gat2Δlpt1Δ, gat1Δslc1Δ, and gat2Δslc1Δ. All mutations mildly reduced [3H]palmitic acid incorporation into phospholipids relative to triacylglycerol. Electrospray ionization tandem mass spectrometry identified few differences from wild type in gat1Δlpt1Δ, dramatic differences in gat2Δslc1Δ, and intermediate changes in gat2Δlpt1Δ and gat1Δslc1Δ. Yeast expressing Gat1p and Lpt1p had phospholipids enriched with acyl chains that were unsaturated, 18 carbons long, and paired for length. These alterations prevented growth at 18.5°C and in 10% ethanol. Therefore, Gat2p and Slc1p dictate phospholipid acyl chain composition in rich media at 30°C. Slc1p selectively pairs acyl chains of different lengths.
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Characterization of a lysophospholipid Acyltransferase involved in membrane remodeling in Candida albicans.
Biochimica et biophysica acta, 2014Co-Authors: Mariam Ayyash, Amal Algahmi, John Gillespie, Peter OelkersAbstract:Abstract Phospholipid remodeling involves phospholipase activity to remove acyl chains and Acyltransferases to replace acyl chains. We here describe the characterization of a lysophospholipid Acyltransferase in the opportunistic fungal pathogen, Candida albicans. Expression of this gene, C.a. LPT1, complemented the lysophospholipid Acyltransferase defect in Saccharomyces cerevisiae strains lacking the homologous LPT1 gene. In vitro, lysophospholipid Acyltransferase activity in these strains showed acyl-CoA substrate specificity, as measured by apparent Vmax/Km ratios, to be linolenoyl-CoA > oleoyl-CoA > linoleoyl-CoA > stearoyl-CoA. To address the physiological importance of C.a. LPT1, homozygous deletion strains were generated. Lysophospholipid Acyltransferase activity with amine containing lysophospholipids was dramatically reduced while lysophosphatidylinositol and lysophosphatidic acid esterification was not significantly lowered. However, C.a. LPT1 over-expression yielded an increased amount of lysophosphatidic Acyltransferase activity, suggesting a role in de novo phospholipid synthesis. LPT1 deletion strains showed slightly slowed growth in standard liquid media but no phenotype in media containing three antifungals that target sterols. To assess the role of C.a. Lpt1 in phospholipid remodeling, an in vivo, pulse–chase assay utilizing polysorbitan palmitate and mass spectrometry was developed. Cellular phospholipid composition became atypical with the provision of palmitate and gradually returned to the typical distribution when palmitate was removed. Deletion of C.a. LPT1 showed a modest yet significant effect on remodeling under these conditions.
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sterol and diacylglycerol Acyltransferase deficiency triggers fatty acid mediated cell death
Journal of Biological Chemistry, 2009Co-Authors: Jeanne Garbarino, Mahajabeen Padamsee, Lisa Wilcox, Diana Dambrosio, Nicole Ramsey, Omar J Jabado, Aaron Turkish, Peter Oelkers, Kelly V Ruggles, Stephen L SturleyAbstract:Deletion of the Acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol Acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast Acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic β-cell dysfunction, diabetes, and the metabolic syndrome.
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a lecithin cholesterol Acyltransferase like gene mediates diacylglycerol esterification in yeast
Journal of Biological Chemistry, 2000Co-Authors: Peter Oelkers, Arthur H Tinkelenberg, Naz Erdeniz, Debra Cromley, Jeffrey T Billheimer, Stephen L SturleyAbstract:Abstract The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian Acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol Acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol Acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in theLRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol Acyltransferase. These mechanisms may also exist in mammalian cells.
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expression in yeast and tobacco of plant cdnas encoding acyl coa diacylglycerol Acyltransferase
FEBS Journal, 2000Co-Authors: Pierrette Bouviernave, Stephen L Sturley, Peter Oelkers, Pierre Benveniste, Hubert SchallerAbstract:During the course of a search for cDNAs encoding plant sterol Acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol Acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol Acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol Acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol Acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2–3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol Acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol Acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol Acyltransferase-transformed plants.
Rosalind A. Coleman - One of the best experts on this subject based on the ideXlab platform.
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analysis of amino acid motifs diagnostic for the sn glycerol 3 phosphate Acyltransferase reaction
Biochemistry, 1999Co-Authors: Tal M. Lewin, Ping Wang, Rosalind A. ColemanAbstract:Alignment of amino acid sequences from various Acyltransferases [sn-glycerol-3-phosphate Acyltransferase (GPAT), lysophosphatidic acid Acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate Acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine Acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I−IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I−IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of Acyltransferases. Using Escherichia coli GPAT (PlsB) as a model Acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I−IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine an...
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triacsin c blocks de novo synthesis of glycerolipids and cholesterol esters but not recycling of fatty acid into phospholipid evidence for functionally separate pools of acyl coa
Biochemical Journal, 1997Co-Authors: R A Igal, P Wang, Rosalind A. ColemanAbstract:The trafficking of acyl-CoAs within cells is poorly understood. In order to determine whether newly synthesized acyl-CoAs are equally available for the synthesis of all glycerolipids and cholesterol esters, we incubated human fibroblasts with [14C]oleate, [3H]arachidonate or [3H]glycerol in the presence or absence of triacsin C, a fungal metabolite that is a competitive inhibitor of acyl-CoA synthetase. Triacsin C inhibited de novo synthesis from glycerol of triacylglycerol, diacylglycerol and cholesterol esters by more than 93%, and the synthesis of phospholipid by 83%. However, the incorporation of oleate or arachidonate into phospholipids appeared to be relatively unimpaired when triacsin was present. Diacylglycerol Acyltransferase and lysophosphatidylcholine Acyltransferase had similar dependences on palmitoyl-CoA in both liver and fibroblasts; thus it did not appear that acyl-CoAs, when present at low concentrations, would be preferentially used to acylate lysophospholipids. We interpret these data to mean that, when fatty acid is not limiting, triacsin blocks the acylation of glycerol 3-phosphate and diacylglycerol, but not the reacylation of lysophospholipids. Two explanations are possible: (1) different acyl-CoA synthetases exist that vary in their sensitivity to triacsin; (2) an independent mechanism channels acyl-CoA towards phospholipid synthesis when little acyl-CoA is available. In either case, the acyl-CoAs available to acylate cholesterol, glycerol 3-phosphate, lysophosphatidic acid and diacylglycerol and those acyl-CoAs that are used by lysophospholipid Acyltransferases and by ceramide N-Acyltransferase must reside in two non-mixing acyl-CoA pools or, when acyl-CoAs are limiting, they must be selectively channelled towards specific Acyltransferase reactions.
Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.
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the wax ester synthase acyl coenzyme a diacylglycerol Acyltransferase from acinetobacter sp strain adp1 characterization of a novel type of Acyltransferase
Journal of Bacteriology, 2005Co-Authors: Tim Stoveken, Rainer Kalscheuer, Ursula Malkus, Rudolf Reichelt, Alexander SteinbuchelAbstract:The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol Acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of Acyltransferases which is fundamentally different from Acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol Acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.
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synthesis of novel lipids in saccharomyces cerevisiae by heterologous expression of an unspecific bacterial Acyltransferase
Applied and Environmental Microbiology, 2004Co-Authors: Rainer Kalscheuer, Heinrich Luftmann, Alexander SteinbuchelAbstract:The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol Acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol Acyltransferase (Lro1p) and two acyl-coenzyme A:sterol Acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific Acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.
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a novel bifunctional wax ester synthase acyl coa diacylglycerol Acyltransferase mediates wax ester and triacylglycerol biosynthesis in acinetobacter calcoaceticus adp1
Journal of Biological Chemistry, 2003Co-Authors: Rainer Kalscheuer, Alexander SteinbuchelAbstract:Abstract Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol Acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol Acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA Acyltransferase is not related to known Acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol Acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatismc2155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinantPseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.