Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.     

Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family

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.     

The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase

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.     

Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT)

BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron–exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals.     

Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters

The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esterified to an alcohol of a similar length. As part of an EST project, we recently identified an acyltransferase with 19% sequence identity (amino acid) to a bacterial ‘bifunctional’ wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deficient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in VLCFA esters of methyl, isoamyl, and short-to-medium straight chain alcohols (C4–C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and ¹⁴C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be sufficient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the first characterization of a eukaryotic protein from the WS/DGAT family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

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.     

Membrane topology and identification of key functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2

Triacylglycerols are the predominant molecules of energy storage in eukaryotes. However, excessive accumulation of triacylglycerols in adipose tissue leads to obesity and, in nonadipose tissues, is associated with tissue dysfunction. Hence, it is of great importance to have a better understanding of the molecular mechanisms of triacylglycerol synthesis. The final step in triacylglycerol synthesis is catalyzed by the acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Although recent studies have shed light on metabolic functions of these enzymes, little is known about the molecular aspects of their structures or functions. Here we report the topology for murine DGAT2 and the identification of key amino acids that likely contribute to enzymatic function. Our data indicate that DGAT2 is an integral membrane protein with both the N and C termini oriented toward the cytosol. A long hydrophobic region spanning amino acids 66-115 likely comprises two transmembrane domains or, alternatively, a single domain that is embedded in the membrane bilayer. The bulk of the protein lies distal to the transmembrane domains. This region shares the highest degree of homology with other enzymes of the DGAT2 family and contains a sequence HPHG that is conserved in all family members. Mutagenesis of this sequence in DGAT2 demonstrated that it is required for full enzymatic function. Additionally, a neutral lipid-binding domain that is located in the putative first transmembrane domain was also required for full enzymatic function. Our findings provide the first insights into the topography and molecular aspects of DGAT2 and related enzymes.     

Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions

The conversion of fatty acids to fatty alcohols is required for the synthesis of wax monoesters and ether lipids. The mammalian enzymes that synthesize fatty alcohols have not been identified. Here, an in silico approach was used to discern two putative reductase enzymes designated FAR1 and FAR2. Expression studies in intact cells showed that FAR1 and FAR2 cDNAs encoded isozymes that reduced fatty acids to fatty alcohols. Fatty acyl-CoA esters were the substrate of FAR1, and the enzyme required NADPH as a cofactor. FAR1 preferred saturated and unsaturated fatty acids of 16 or 18 carbons as substrates, whereas FAR2 preferred saturated fatty acids of 16 or 18 carbons. Confocal light microscopy indicated that FAR1 and FAR2 were localized in the peroxisome. The FAR1 mRNA was detected in many mouse tissues with the highest level found in the preputial gland, a modified sebaceous gland. The FAR2 mRNA was more restricted in distribution and most abundant in the eyelid, which contains wax-laden meibomian glands. Both FAR mRNAs were present in the brain, a tissue rich in ether lipids. The data suggest that fatty alcohol synthesis in mammals is accomplished by two fatty acyl-CoA reductase isozymes that are expressed at high levels in tissues known to synthesize wax monoesters and ether lipids.     

Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members

Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT, DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol, indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity was inhibited by a high concentration (100 mm) of MgCl(2) in an in vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.     

Physiological characterization of lipid accumulation and in vivo ester formation in Gordonia sp. KTR9

Previous work has demonstrated the feasibility of in vivo biodiesel synthesis in Escherichia coli, however, ethyl ester formation was dependent on an external fatty acid feedstock. In contrast to E. coli, actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). In this study, we investigated the physiology and associated TAG accumulation along with the in vivo ability to catalyze ester formation from exogenous short chain alcohol sources in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Strain KTR9 tolerated the exogenous addition of up to 4 % methanol, 4 % ethanol and 2 % propanol in the media. Increasing alcohol concentrations resulted in a decrease in the degree of saturation of recovered fatty acid alcohol esters and a slight increase in the fatty acid chain length. A linear dose dependency in fatty alcohol ester synthesis was observed in the presence of 0.5–2 % methanol and ethanol compared to control KTR9 strains grown in the absence of alcohols. An inspection of the KTR9 genome revealed the presence of several putative wax ester synthase/acyl-coenzyme A?:?diacylglycerol acyltransferase (WS/DGAT) enzymes, encoded by atf gene homologs, that may catalyze the in vivo synthesis of fatty acid esters from short chain alcohols. Collectively, these results indicate that Gordonia sp. KTR9 may be a suitable actinomycete host strain for in vivo biodiesel synthesis.     

Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases

Marinobacter hydrocarbonoclasticus DSM 8798 has been reported to synthesize isoprenoid wax ester storage compounds when grown on phytol as the sole carbon source under limiting nitrogen and/or phosphorous conditions. We hypothesized that isoprenoid wax ester synthesis involves (i) activation of an isoprenoid fatty acid by a coenzyme A (CoA) synthetase and (ii) ester bond formation between an isoprenoid alcohol and isoprenoyl-CoA catalyzed, most likely, by an isoprenoid wax ester synthase similar to an acyl wax ester synthase, wax ester synthase/diacylglycerol acyltransferase (WS/DGAT), recently described from Acinetobacter sp. strain ADP1. We used the recently released rough draft genome sequence of a closely related strain, M. aquaeolei VT8, to search for WS/DGAT and acyl-CoA synthetase candidate genes. The sequence information from putative WS/DGAT and acyl-CoA synthetase genes identified in this strain was used to clone homologues from the isoprenoid wax ester synthesizing Marinobacter strain. The activities of the recombinant enzymes were characterized, and two new isoprenoid wax ester synthases capable of synthesizing isoprenoid ester and acyl/isoprenoid hybrid ester in vitro were identified along with an isoprenoid-specific CoA synthetase. One of the Marinobacter wax ester synthases displays several orders of magnitude higher activity toward acyl substrates than any previously characterized acyl-WS and may reflect adaptations to available carbon sources in their environments.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.     

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1

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. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.     

Inhibition of diacylglycerol acyltransferase by 2-bromooctanoate in cultured rat hepatocytes

Triacylglycerol synthesis in cultured rat hepatocytes was inhibited by 2-bromooctanoate with a concomitant accumulation of diacylglycerols. 2-Bromooctanoate inhibition could be ascribed to its thioesterification by medium chain fatty acyl-CoA synthase (Raaka, B.M., and Lowenstein, J.M. (1979) J. Biol. Chem. 254, 6755-6762) with 2-bromooctanoyl-CoA acting as a competitive inhibitor of diacylglycerol acyltransferase. The Ki of 2-bromooctanoyl-CoA was 1.5 microM compared with a Km of 25 microM for the palmitoyl-CoA substrate. Diacylglycerol esterification was also inhibited by C12-C16 2-bromo fatty acids. However, inhibition of triacylglycerol synthesis by long chain 2-bromo fatty acids was accompanied by decreased overall neutral lipid synthesis as a result of inhibition of the long chain fatty acyl-CoA synthase. Since 2-bromooctanoate was a poor inhibitor of the long chain fatty acyl-CoA synthase, it appears to function selectively as an inhibitor of diacylglycerol acyltransferase in cultured rat hepatocytes.     

Molecular and Functional Analysis of Three Fatty Acyl-CoA Reductases with Distinct Substrate Specificities in Copepod Calanus finmarchicus

The marine copepod Calanus finmarchicus constitutes the substantial amount of biomass in the Arctic and Northern seas. It is unique in that this small crustacean accumulates a high level of wax esters as carbon storage which is mainly comprised of 20:1n−9 and 22:1n−11 alcohols (Alc) linked with various kinds of fatty acids, including n−3 polyunsaturated fatty acids. The absence of 20:1n−9 Alc and 22:1n−11 Alc in diatoms and dinoflagellates, the primary food sources of copepods, suggests the existence of de novo biosynthesis of fatty alcohols in C. finmarchinus. Here, we report identification of three genes, CfFAR1, CfFAR2, and CfFAR3, coding for fatty acyl-CoA reductases involved in the conversion of various fatty acyl-CoAs to their corresponding alcohols. Functional characterization of these genes in yeast indicated that CfFAR1 could use a wide range of saturated fatty acids from C18 to C26 as substrates, CfFAR2 had a narrow range of substrates with only very-long-chain saturated fatty acid 24:0 and 26:0, while CfFAR3 was active towards both saturated (16:0 and 18:0) and unsaturated (18:1 and 20:1) fatty acids producing corresponding alcohols. This finding suggested that these three fatty acyl-CoA reductases are likely responsible for de novo synthesis of a series of fatty alcohol moieties of wax esters in C. finmarchicus.     

Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase

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.     

ELOVL2 overexpression enhances triacylglycerol synthesis in 3T3-L1 and F442A cells

Elongation of very long-chain fatty acids (ELOVL) members were overexpressed in two preadipocyte cell lines, ELOVL2 and ELOVL3 in 3T3-L1 cells, and ELOVL1-3 in F442A cells. Cells overexpressing ELOVL2, whose preferred substrates are arachidonic acid (AA, C20:4n-6) and eicosapentaenoic acid (EPA, C20:5n-3), showed an enhanced triacylglycerol (TAG) synthesis and subsequent accumulation of lipid droplets. Incorporation of fatty acid (FA) but not of glucose into TAG was enhanced by ELOVL2-overexpression. Two lipogenic genes encoding diacylglycerol acyltransferase-2 (DGAT2) and fatty acid-binding protein-4 (FABP4, aP2) were induced in ELOVL2-overexpressing cells, whereas no such effect was seen on the fatty acid synthase (FAS) gene.     

Isolation of a gene encoding a 1,2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus

1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alatus (Burning Bush). These ac-TAGs have long-chain acyl groups esterified at both the sn-1 and sn-2 positions of glycerol. Cell-free extracts of developing seeds of E. alatus contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity. We have isolated a gene from developing seeds of Euonymus alatus that shows a very high sequence similarity to the members of the DGAT1 gene family (i.e. related to acyl-CoA:cholesterol acyltransferases). This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of long-chain triacylglycerol (lc-TAG) accumulation, as well as the appearance of low levels of ac-TAG. Hydrogenated ac-TAG molecular species were identified by gas chromatography-mass spectrometry. Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity, which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DGAT1 gene. The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector or either the Arabidopsis or Euonymus DGAT1 genes. Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGAT1 gene was very low (0.26% of lc-TAG), whereas lc-TAG accumulation was enhanced. Possible reasons for this anomaly are discussed. Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG synthesis has been deleted confirmed that the gene product has both long-chain acyl- and acetyltransferase activity.     

Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase.

Acinetobacter calcoaceticus BD413 accumulates wax esters and triacylglycerol under conditions of mineral nutrient limitation. Nitrosoguanidine-induced mutants of strain BD413 were isolated that failed to accumulate wax esters under nitrogen-limited growth conditions. One of the mutants, Wow15 (without wax), accumulated wax when grown in the presence of cis-11-hexadecenal and hexadecanol but not hexadecane or hexadecanoic acid. This suggested that the mutation may have inactivated a gene encoding either an acyl-acyl carrier protein or acyl-coenzyme A (CoA) reductase. The Wow15 mutant was complemented with a cosmid genomic library prepared from wild-type A. calcoaceticus BD413. The complementary region was localized to a single gene (acr1) encoding a protein of 32,468 Da that is 44% identical over a region of 264 amino acids to a product of unknown function encoded by an open reading frame associated with mycolic acid synthesis in Mycobacterium tuberculosis H37Ra. Extracts of Escherichia coli cells expressing the acr1 gene catalyzed the reduction of acyl-CoA to the corresponding fatty aldehyde, indicating that the gene encodes a novel fatty acyl-CoA reductase.     

Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) is a membrane protein present mainly in the endoplasmic reticulum. It catalyzes the final and committed step in the biosynthesis of triacylglycerol, which is the principal repository of fatty acids for energy utilization and membrane formation. Two distinct family members of acyl-CoA:diacylglycerol acyltransferase, known as DGAT1 and DGAT2, have been characterized in different organisms, including mammals, fungi, and plants. In this study, we characterized the functional role and topological orientation of signature motifs in yeast (Saccharomyces cerevisiae) DGAT2 using mutagenesis in conjunction with chemical modification. Our data provide evidence that both the N and C termini are oriented toward the cytosol and have different catalytic roles. A highly conserved motif, (129)YFP(131), and a hydrophilic segment exclusive to yeast DGAT2 reside in a long endoplasmic reticulum luminal loop following the first transmembrane domain and play an essential role in enzyme catalysis. In addition, the strongly conserved His(195) within the motif HPHG, which may play a role in the active site of DGAT2, is likely embedded in the membrane. These results indicate some similarities to the topology model of murine DGAT2 but also reveal striking differences suggesting that the topological organization of DGAT2 is not ubiquitously conserved.