Microsomal Desaturase Research Articles

Molecular cloning and characterization of the genes encoding a microsomal oleate Δ12 desaturase (CsFAD2) and linoleate Δ15 desaturase (CsFAD3) from Camelina sativa

Camelina sativa produces oil that is rich in polyunsaturated linoleic (18:2Δ9,12) and linolenic (18:3Δ9,12,15) acids. These fatty acids are obtained by the successive desaturation of oleic (18:1Δ9) acid, which is catalyzed in the endoplasmic reticulum by two different microsomal desaturases: oleate Δ12 desaturase (FAD2) and linoleate Δ15 desaturase (FAD3). The objective of the present study is to investigate the contribution of these two desaturases to the composition and properties of C. sativa seed oil, and look over the effect that temperature exerts on their activity. Three different copies of both these genes were identified, which when analysed contained three histidine rich motifs (HXCGHX, HRXHH and HVXHH) and six highly conserved transmembrane domains. Comparing their sequences, the CsFAD2 copies accommodated four conservative changes (Glu-36-Asp, Arg-48-His, Val-97-Ala and Ala-177-Pro) and two semi-conservative ones (Val-63-Ile and Leu-249-Met), whereas only one semi-conservative change (Ala-327-Ser) was detected in CsFAD3 but with two extra amino acids (His-147 and Gly-148). The CsFAD2 and CsFAD3 cDNAs were heterologously expressed in Saccharomyces cerevisiae to confirm that they were active enzymes and their dependence on temperature was investigated by growing the recombinant yeast cells at low (22°C) and optimal (30°C) temperatures. The conversion of 18:1Δ9 to 18:2Δ9,12 by CsFAD2 was slightly better at 30°C than at 22°C, and it was also able to desaturate palmitoleic acid (16:1Δ9) to hexadecadienoic acid (16:2Δ9,12). By contrast, the conversion driven by CsFAD3 was enhanced 5-fold at 22°C compared to 30°C, although it was not able to desaturate 16:2Δ9,12. The distribution of these microsomal desaturases was also studied in C. sativa, which were expressed most strongly in expanding leaves and developing seeds.

Differential transcriptional activity of SAD, FAD2 and FAD3 desaturase genes in developing seeds of linseed contributes to varietal variation in α-linolenic acid content

Linseed or flax (Linum usitatissimum L.) varieties differ markedly in their seed α-linolenic acid (ALA) levels. Fatty acid desaturases play a key role in accumulating ALA in seed. We performed fatty acid (FA) profiling of various seed developmental stages of ten Indian linseed varieties including one mutant variety. Depending on their ALA contents, these varieties were grouped under high ALA and low ALA groups. Transcript profiling of six microsomal desaturase genes (SAD1, SAD2, FAD2, FAD2-2, FAD3A and FAD3B), which act sequentially in the fatty acid desaturation pathway, was performed using real-time PCR. We observed gene specific as well as temporal expression pattern for all the desaturases and their differential expression profiles corresponded well with the variation in FA accumulation in the two groups. Our study points to efficient conversion of intermediate FAs [stearic (SA), oleic (OA) and linoleic acids (LA)] to the final product, ALA, due to efficient action of all the desaturases in high ALA group. While in the low ALA group, even though the initial conversion up to OA was efficient, later conversions up to ALA seemed to be inefficient, leading to higher accumulation of OA and LA instead of ALA. We sequenced the six desaturase genes from the ten varieties and observed that variation in the amino acid (AA) sequences of desaturases was not responsible for differential ALA accumulation, except in the mutant variety TL23 with very low (<2%) ALA content. In TL23, a point mutation in the FAD3A gene resulted into a premature stop codon generating a truncated protein with 291 AA.

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.

Dimethyl acetals, an indirect marker of the endogenous antioxidant plasmalogen level, are reduced in blood lipids of Sudanese pre-eclamptic subjects whose background diet is high in carbohydrate

In Sudanese women with (n = 60) and without (n = 65) pre-eclampsia, circulating lipids, plasma and red cell saturated and monounsaturated fatty (MUFA) acids and dimethyl acetals (DMAs) were investigated. DMAs are an indirect marker of levels of plasmalogens, endogenous antioxidants, which play a critical role in oxidative protection, and cholesterol homeostasis. The pre-eclamptics had higher C18:1n-9 (p < 0.001) and ΣMUFA (p < 0.01) in plasma free fatty acids, C16:1n-7, C18:1n-9, ΣMUFA; 16:0/16:1n-7 (p < 0.01) in erythrocyte choline phosphoglycerides (ePC) and 16:1n-7, 18:1n-7 and 16:0/16:1n-7 (p < 0.01) in erythrocyte ethanolamine phosphoglycerides (ePE). In contrast, the DMAs 18:0, 18:1 and ΣDMAs in ePE, and 16:0, 18:0 and ΣDMAs in ePC were reduced (p < 0.001) in the pre-eclamptic women. This study of pregnant women with high carbohydrate and low fat background diet suggests pre-eclampsia is associated with oxidative stress and enhanced activity of the microsomal enzyme stearyl-CoA desaturase (delta 9 desaturase), as assessed by palmitic/palmitoleic (C16:0/C16:n-1) and stearic/oleic (C18/C18:1n-9) ratios.

The sunflower plastidial ω3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity

Although plant plastidial omega3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 omega3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 omega3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.

Cloning, Tissue Expression Analysis, and Functional Characterization of Two Δ6-Desaturase Variants of Sea Bass (Dicentrarchus labrax L.)

Fish are the main source of the n-3 highly unsaturated fatty acids, which are crucial for human health. Their synthesis from C(18) precursors is mediated by desaturases and elongases, but the activity of these enzymes has not been conclusively established in marine fish species. This study reports the cloning, tissue expression, and functional characterization of a sea bass (Dicentrarchus labrax L.) Δ6-desaturase and one of its splicing variants. Two cDNAs with open reading frames of 1,346 and 1,354 bp were cloned and named D6D and D6D-V, respectively. Both deduced protein sequences (445 and 387 amino acids, respectively) contained two transmembrane regions and the N-terminal cytochrome b(5) domain with the HPGG motif characteristic of microsomal desaturases. D6D presents three histidine-rich regions, whereas in D6D-V, an insertion of eight nucleotides in the boundaries of exons 10 and 11 modified the third histidine-rich domain and led to insertion of a premature STOP codon, resulting in a shorter predicted protein. Quantitative real-time polymerase chain reaction assay of gene expression showed that D6D was highly expressed in the brain and intestine, and to a lesser extent, in muscle and liver; meanwhile, D6D-V was expressed in all tissues tested, but at level at least 200-fold lower than D6D. Functional analysis in yeast showed that sea bass D6D encodes a fully functional Δ6-desaturase with no residual Δ5-desaturase activity. This desaturase does not exhibit a clear preference for n-3 versus n-6 C(18) substrates. Interestingly, D6D-V is a nonfunctional protein, suggesting that the C-terminal end is indispensable for protein activity.

Fatty acids of plant vacuolar membrane lipids

Fatty acid (FA) composition of vacuolar membrane lipids from storage tissues of umbelliferous plants, viz., parsnip (Pastinaca sativa L.), parsley (Petroselinium crispum L.), and carrot (Daucus carota L.) is studied by gas-liquid chromatography and the FA biosynthetic pathways are considered. Vacuolar membrane lipids are characterized by high (78% of the total FA pool) content of unsaturated FA among which linoleic acid is predominant. Its content in vacuolar lipids of parsnip, parsley and carrot is 53.5, 55.1, and 54.9%, respectively. Parsnip and parsley vacuolar lipids contain large amounts of hexadecadienoic C16:2ω6 acid (8.0 and 4.6%, respectively). The content of α-linolenic acid in vacuolar lipids of tested plants varies from 4.8 to 7.3%. Palmitic acid (18.0–20.7%) predominates among saturated FA. High content of linoleic and hexadecadienoic acid in parsnip and parsley vacuolar lipids is suggestive of a crucial role of the microsomal ω6 fatty-acid desaturase fad2 gene in resistance and acclimation of plants to low temperatures.

Regulation of desaturase gene expression, changes in membrane lipid composition and freezing tolerance in potato plants

Irreversible damage of the plant plasma membrane is the primary cause of freezing injury. Changes in lipid unsaturation have being largely studied as one of the major components aimed at preserving the integrity and functionality of plasma membranes, and increasing freezing tolerance. In the present study, the potato stearoyl-ACP desaturase (ω-9) gene, encoding the first enzyme involved in plant lipid unsaturation, was cloned and used to monitor its expression during cold acclimation in plants of two Solanum species, known to differ in their ability to cold acclimate. Although up-regulated upon cold acclimation in both potato species, freezing tolerant S. commersonii plants had a constitutive level of the ω-9 desaturase gene transcripts and of other known stress protective proteins (dhn2 and cpn60β) remarkably higher than plants of the not freezing tolerant potato species (S. tuberosum). Transcript levels of oleoyl-desaturase (ω-6) and linoleyl CoA desaturase (ω-3), microsomal desaturases involved in further plasma membrane fatty acid (FA) unsaturation, did not vary appreciably during cold acclimation in both potato species. S. tuberosum potato plants overexpressing the ω-9 desaturase gene were generated, to change FA lipid composition and measure the effect on the basal level freezing tolerance of cultivated potato varieties. Unsaturation level of total leaf polar lipids of one of best ω-9 desaturase overexpressing line, with a high transcript level of the exogenous gene and related protein, was slightly higher compared to potato plants transformed with the empty vector, with the notable appearance of cis-vaccenic acid (C18:1 Δ11), an unusual monoic FAs in plants. Freezing tolerance, estimated as an increase in LT50 (2°C), derived from the electrolyte leakage test, enhanced in ω-9 desaturase overexpressing transgenic potato plants only upon cold acclimation. These results indicate that modifications in lipid unsaturation account for only a small fraction of the acquired freezing tolerance, while interaction with other protective proteins (dhn2, cpn60β) is necessary to fulfil a higher level of freezing tolerance.

A Bifunctional Δ12,Δ15-Desaturase from Acanthamoeba castellanii Directs the Synthesis of Highly Unusual n-1 Series Unsaturated Fatty Acids

The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.

Solid-State NMR Studies of a Diverged Microsomal Amino-Proximate Δ12 Desaturase Peptide Reveal Causes of Stability in Bilayer: Tyrosine Anchoring and Arginine Snorkeling

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal Δ12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L α phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8° with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.

Two FAD3 desaturase genes control the level of linolenic acid in flax seed.

Industrial oil flax (Linum usitatissimum) and edible oil or solin flax differ markedly in seed linolenic acid levels. Despite the economic importance of low-linolenic-acid or solin flax, the molecular mechanism underlying this trait has not been established. Two independently inherited genes control the low-linolenic-acid trait in flax. Here, we identified two genes, LuFAD3A and LuFAD3B that encode microsomal desaturases capable of desaturating linoleic acid. The deduced proteins encoded by these genes shared 95.4% identity. In the low-linolenic-acid line solin 593-708, both LuFAD3A and LuFAD3B carry point mutations that produce premature stop codons. Expression of these genes in yeast (Saccharomyces cerevisiae) demonstrated that, while the wild-type proteins were capable of desaturating linoleic acid, the truncated proteins were inactive. Furthermore, the low-linolenic-acid phenotype in flax was complemented by transformation with a wild-type gene. Codominant DNA markers were developed to distinguish between null and wild-type alleles of both genes, and linolenic acid levels cosegregated with genotypes, providing further proof that LuFAD3A and LuFAD3B are the major genes controlling linolenic acid levels in flax. The level of LuFAD3 transcripts in seeds peaked at about 20 d after flowering, and transcripts were not detectable in leaf, root, or stem tissue. A dramatic reduction in transcript levels of both genes occurred in the low-linolenic-acid solin line, which was likely due to nonsense-mediated decay.

Spatial and Temporal Expression of Mutations for High Oleic Acid and Low Linolenic Acid Concentration in Ethiopian Mustard

The development of Ethiopian mustard (Brassica carinata A. Braun) germplasm with high concentration of oleic acid and low concentration of linolenic add in its seed oil is an important breeding objective in this crop. The mutant AB-1, with high oleic acid and low linolenic acid concentration, and the mutant AB-4, with very low linolenic add concentration have recently been developed. In both cases, mutations are expressed at the seed oil level, but their level of expression in other plant tissues has not been studied. The objective of the present research was to monitor the expression of altered fatty acid profiles in AB-1 and AB-4 in different plant tissues and stages of seed development and germination. Plants of AB-1 expressed the high oleic acid trait in pollen, roots, and leaves and the low linolenic add trait in pollen and roots. Plants of the AB-4 mutant expressed the low linolenic add trait in pollen, roots, and leaves. The high oleic acid trait of AB-1 was expressed during the whole seed developing period, and also in 10-d-old cotyledons. The low linolenic acid trait of AB-4 was expressed during seed development and also from 0 to 8 d after germination. The expression of mutations for high oleic add and low linolenic acid in Ethiopian mustard mutants AB-1 and AB-4 in plant tissues other than seeds may have important breeding implications. Comparison of these results with previous studies on Brassica napus L. and Arabidopsis mutants suggested that AB-1 might show altered activity at both FAD2 and FAD3 microsomal desaturases, whereas AB-4 might show reduced activity at FAD3 level.

Developmental Expression of Microsomal Omega-6 Desaturase Gene (fad2-1) in Soybean Seeds

Fad2-1 gene encoded microsomal omega-6 desaturase plays a major role in controlling the conversion of oleic acid to linoleic acid within storage lipids during seed development. A 180 bp region (1241–1422) specific to fad2-1 was amplified and its sequence confirmed. Northern blot analysis using the labelled amplicon as probe indicated that the fad2-1 expression was induced during early stages of embryo development and peaked in the mid-maturation stages. Tissue specificity of fad2-1 transcripts was confirmed by the complete absence of fad2-1 transcripts in the vegetative tissues of leaf, stem and root. Developmental profile of fatty acids showed increased levels of linoleic acid during the mid-maturation stages which coincided with the enhanced expression of fad2-1 during the same period.

New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases.

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.

Isolation and characterization of a cDNA encoding a Delta8 sphingolipid desaturase from Aquilegia vulgaris.

We have isolated a cDNA encoding the Delta(8) sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis - and trans -desaturated sphingoid bases which were not present in the wild-type yeast cells. The Delta(8) desaturated products co-eluted with known Delta(8)-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Delta(8) long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.

Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

The Δ8-Desaturase ofEuglena gracilis:An Alternate Pathway for Synthesis of 20-Carbon Polyunsaturated Fatty Acids

Desaturation of fatty acids is an important metabolic process. In mammals, 20-carbon and longer polyunsaturated fatty acids are not only incorporated into cellular membranes in a tissue-specific manner, but also serve as the precursors to synthesis of eicosanoid metabolic regulators. The processes of desaturation and elongation in human liver are well characterized, but an alternate Δ8desaturation pathway that may be important in certain tissues or in cancer cells is less well examined. The Δ8-desaturase enzyme introduces a double bond at the 8-position in 20-carbon fatty acids that have an existing Δ11 unsaturation. We have isolated the first fatty acid Δ8-desaturase, from the protistEuglena gracilis,in order to explore this alternate pathway. A full-length cDNA was obtained after reverse transcription of mRNA purified from heterotrophically grownEuglena,followed by PCR amplification with primers degenerate to conserved histidine-rich regions of microsomal desaturases. The protein predicted from the cDNA sequence is highly homologous to Δ5and Δ6desaturases ofCaenhorabditis elegans.When the cDNA was expressed inSaccharomyces cerevisiae,the yeast cultures readily desaturated appropriate 20-carbon fatty acids by inserting an additional double bond at the Δ8-position. The enzyme demonstrated a preference for substrates of metabolic significance, 20:3 Δ11,14,17 and 20:2 Δ11,14. Cloning of a Δ8fatty acid desaturase offers the opportunity to examine an alternate pathway of long chain fatty acid biosynthesis.

Characterization and expression of a fatty acid desaturase from Borago officinalis

accumulated in the leaves, indicating that the cDNA encoded A homologue of the FAD2 microsomal D12-fatty acid desatur- a microsomal D6-fatty acid desaturase (Sayanova et al., ase was isolated from borage (Borago officinalis), a species 1997). This D6-fatty acid desaturase was unlike any previnoted for the synthesis of unusual fatty acids. Expression of ously characterized higher plant microsomal desaturases, due the borage FAD2 transcript was correlated with the levels of to the presence of an N-terminal extension of 100 amino linoleic acid, and expression of this FAD2 homologue was acids, which showed homology to the electron transport highest in non-photosynthetic tissues. chain protein cytochrome b 5 (Napier et al., 1997). The presence of this N-terminal extension has subsequently been

Isolation of a Δ5-Fatty Acid Desaturase Gene fromMortierella alpina

Arachidonic acid (C20:4 Delta5,8,11,14) is a polyunsaturated fatty acid synthesized by the Delta5-fatty acid desaturation of di-homo-gamma-linolenic acid (C20:3 Delta8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Delta5-fatty acid desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Delta5-desaturase is the first example of a cloned Delta5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b5.

Degradation of hepatic stearyl CoA delta 9-desaturase.

delta 9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt-washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.