Identification of an animal ω-3 fatty acid desaturase by heterologous expression in 
            <i>Arabidopsis</i>

Spychalla, James P.; Kinney, Anthony J.; Browse, John

doi:10.1073/pnas.94.4.1142

Cited by 204 publications

(163 citation statements)

References 32 publications

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“…Previously, Browse and coworkers (27) functionally characterized a C. elegans 3 -fatty acid desaturase with specificity for both C18 and C20 substrates that was capable of producing 20:5, n-3 from 20:4, n-6. Thus, the biosynthetic pathway for 20:5, n-3 could be via either a final methyl-directed desaturation of 20:4 n-6 or the ⌬ 5 -desaturation of the 20:4, n-3 fatty acid.…”

Section: Discussionmentioning

confidence: 99%

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway

Beaudoin

Michaelson

Hey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

114

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A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11.4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for ⌬ 6 -desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from ␣-linolenic acid. These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways. The C. elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates.

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Section: Discussionmentioning

confidence: 99%

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway

Beaudoin

Michaelson

Hey

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

114

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“…Populations derived from individuals of the first and third class always had fatty acid compositions identical to their parents, whereas the progeny of the second class fell into all three classes, which indicates that the reduction in n3 fatty acids was caused by a single nuclear mutation, and that members of the third class were homozygous for the mutant allele. Because no n3 PUFAs were produced in the homozygous lines and the n6 precursors were increased, we hypothesized that the mutation affected the fat-1 locus, which encodes a gene product that has been shown to possess n3 desaturase activity (8,21). To test this hypothesis, RNA from the mutant strains was isolated and the coding region of the fat-1 gene was amplified by reverse transcription-PCR and directly sequenced.…”

Section: Fat-1 Mutants Lack N3mentioning

confidence: 99%

“…To this end, C. elegans expresses the full range of desaturase activities found in plants (⌬12 and n3 desaturase) and animals (⌬5 and ⌬6 desaturase) as well as n6 and n3 PUFA elongase activities found in animals. cDNAs corresponding to C. elegans n3, ⌬12, ⌬6, and ⌬5 desaturase genes have been expressed in Arabidopsis and yeast to confirm their desaturase activity (8)(9)(10)(11). Another C. elegans gene confers the ability to elongate ␥-linolenic acid (18:3n6) and stearidonic acid (18:4n3) when expressed in yeast (12).…”

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confidence: 99%

Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans

Watts

Browse

2002

Proc. Natl. Acad. Sci. U.S.A.

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370

425

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Polyunsaturated fatty acids (PUFAs) are important membrane components and precursors of signaling molecules. To investigate the roles of these fatty acids in growth, development, and neurological function in an animal system, we isolated Caenorhabditis elegans mutants deficient in PUFA synthesis by direct analysis of fatty acid composition. C. elegans possesses all the desaturase and elongase activities to synthesize arachidonic acid and eicosapentaenoic acid from saturated fatty acid precursors. In our screen we identified mutants with defects in each fatty acid desaturation and elongation step of the PUFA biosynthetic pathway. The fatty acid compositions of the mutants reveal the substrate preferences of the desaturase and elongase enzymes and clearly demarcate the steps of this pathway. The mutants show that C. elegans does not require n3 or ⌬5-unsaturated PUFAs for normal development under laboratory conditions. However, mutants with more severe PUFA deficiencies display growth and neurological defects. The mutants provide tools for investigating the roles of PUFAs in membrane biology and cell function in this animal model. P olyunsaturated fatty acid (PUFA) components of phospholipids are necessary to create a fluid membrane environment. In mammals, specific PUFA composition affects many cellular processes including modulation of ion channels (1, 2), endocytosis͞exocytosis (3), and activities of membrane-associated enzymes that are sensitive to the biophysical properties of lipid membranes (4). In addition, certain classes of PUFAs are precursors of powerful eicosanoid effectors such as prostaglandins and leukotrienes (5). Disruptions in proper PUFA intake and metabolism are associated with certain human disease states such as coronary artery disease, hypertension, diabetes, inflammatory disorders, and cancer (6). In most cases the mechanisms through which fatty acid composition affects these conditions are not well understood.Mammals require the essential fatty acids linoleic acid (18:2n6) and linolenic acid (18:3n3) in their diet. Desaturation and elongation of these fatty acids produce the C20 PUFAs that become membrane components and precursors for eicosanoids. Plants produce these essential n6 and n3 PUFAs by desaturating oleic acid (18:1n9) at C12 and C15 positions to produce 18:2n6 and 18:3n3 but rarely possess the enzymes necessary to desaturate and elongate them further into C20 PUFAs. The free-living nematode Caenorhabditis elegans synthesizes a wide range of PUFAs including arachidonic acid (20:4n6) and eicosapentaenoic acid (20:5n3) by using saturated fatty acids obtained from their Escherichia coli diet (7). To this end, C. elegans expresses the full range of desaturase activities found in plants (⌬12 and n3 desaturase) and animals (⌬5 and ⌬6 desaturase) as well as n6 and n3 PUFA elongase activities found in animals. cDNAs corresponding to C. elegans n3, ⌬12, ⌬6, and ⌬5 desaturase genes have been expressed in Arabidopsis and yeast to confirm their desaturase activity (8-11). Another C. elegans ...

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“…The "⌬12" (FAT-2) and -3 (FAT-1) fatty acid desaturases from the nematode Caenorhabditis elegans were used as a model system. These enzymes are involved in the biosynthesis of polyunsaturated fatty acids in C. elegans that range from 18:2(9,12) to 20:5 (5,8,11,14,17) (17)(18)(19)(20). The two enzymes share 51% amino acid sequence identity.…”

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confidence: 99%

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

Sasata

Reed

Loewen

et al. 2004

Journal of Biological Chemistry

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Most fatty acid desaturases are members of a large superfamily of integral membrane, O 2 -dependent, ironcontaining enzymes that catalyze a variety of oxidative modifications to lipids. Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification. To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans -3 (FAT-1) and "⌬12" (FAT-2) desaturases were used as a model system. With the use of unnatural substrates, the regioselectivity of C. elegans FAT-2 was clearly defined as ؉3, i.e. it "measures" three carbons from an existing double bond. The structural basis for ؉3 and -3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2. Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase. When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from ؉3 to -3 and vice versa. In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes and the carboxyl-terminal region.Fatty acid desaturases are part of multicomponent systems that catalyze the oxygen-and nicotinamide adenine dinucleotidedependent syn-dehydrogenation of unactivated aliphatic regions of their fatty ester (acyl-lipid) or thioester (acyl-acyl carrier protein or acyl-CoA) substrates (1-6). In addition to the family of soluble fatty acid desaturases, of which the plant stearoyl-acyl carrier protein desaturase is well characterized, there is a large group of structurally distinct integral membrane desaturases. Membrane desaturases of widely varying substrate specificity and regioselectivity are scattered among a range of taxa. The yeast Saccharomyces cerevisiae has a single stearoyl-CoA ⌬9 desaturase, whereas many bacteria lack such desaturases entirely. At the other extreme, the biosynthesis of (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid in some marine fungi likely requires the successive action of six structurally similar membrane desaturases, each varying in substrate specificity and regioselectivity (7,8).Based on structural and catalytic similarities, membrane desaturases are included in a superfamily of oxidative enzymes along with alkane hydroxylase, xylene monooxygenase, carotene ketolase, and sterol methyloxidase (9, 10). These are thought to contain a histidine-coordinated diiron center at the active site. While essentially no three-dimensional structural information is available for these difficult to purify enzymes, primary structure similarity, especially the conservation of three histidine-rich motifs, and similarity o...

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Identification of an animal ω-3 fatty acid desaturase by heterologous expression in Arabidopsis

Cited by 204 publications

References 32 publications

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway

Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

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