Molecular Analysis of a Bifunctional Fatty Acid Conjugase/Desaturase from Tung. Implications for the Evolution of Plant Fatty Acid Diversity

Abstract

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% ␣-eleostearic acid (18: 3⌬ 9cis,11trans,13trans ), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related ⌬ 12 oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1⌬ 9cis ) into linoleic acid (18:2⌬ 9cis,12cis ) and that FADX converted linoleic acid into ␣-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and ⌬ 12 -desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with ⌬ 12 fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.Conjugated fatty acids are naturally occurring compounds that have specialized uses in nutraceutical and industrial applications. For example, conjugated linoleic acid (CLA) is a potent anticancer compound present in foods derived from ruminant animals (Belury, 2002). This bioactive fatty acid (predominantly the 18:2⌬ 9cis,11trans isomer) is synthesized by rumen bacteria and then absorbed by the animal and concentrated in milk fat or adipose tissue. Rumen bacteria also synthesize 18:1⌬ 11trans , which can be absorbed and then desaturated by an animal stearoyl-CoA desaturase to produce CLA (Corl et al., 2001). Conjugated fatty acids such as ␣-eleostearic acid (18:3⌬ 9cis,11trans,13trans ) have recently shown promise for anticancer applications (Igarashi and Miyazawa, 2000;Kohno et al., 2002), as well as serum lipidlowering effects in mammals (Koba et al., 2002). Oils containing ␣-eleostearic acid may also be used for industrial drying applications. Tung oil, which is derived from seeds of the tung tree (Aleurites fordii Hemsl.), is commonly used in formulations of inks, dyes,...

Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria

“…Figure 1D shows also that coexpression of any of the four tung Cb5 proteins with FAD2 in the mutant DCYB5 strain fully restored FAD2 activity, demonstrating that each Cb5 protein could functionally complement yeast Cb5 activity. Similar complementation results were obtained when using N-terminally myc-tagged Cb5 proteins or when each Cb5 protein was coexpressed in DCYB5 cells with a different Cb5-dependent FAD, including tung FADX (Dyer et al, 2002) (data not shown).…”

“…(D) Functional complementation of yeast Cb5 protein activity. Wild-type or mutant (MUT; lacking the endogenous CYB5 gene) yeast cells were either transformed with FAD2 (a Cb5-dependent desaturase that produces linoleic acid from endogenous oleic acid; Dyer et al, 2002) or cotransformed with FAD2 and either tung Cb5-A, -B, -C, or -D. Strain and plasmid combinations are shown along the x axis. Cells were grown overnight in galactose-containing medium and then harvested, lipids were extracted, and fatty acid composition determined by gas chromatography and flame ionization detection using methyl heptadecanoate as an internal standard.…”

“…To assess the functional properties of the four tung Cb5 proteins, each isoform was expressed individually in yeast cells that harbored a disruption in the single endogenous Cb5 gene (CYB5) but contained an ectopically expressed, Cb5-dependent FAD2 from tung (Dyer et al, 2002). Control experiments revealed that FAD2 activity was substantially reduced in mutant cells lacking the endogenous Cb5 protein, as judged by a decrease in the amount of linoleic acid produced by FAD2 in DCYB5 cells in comparison with the amount produced by FAD2 in wild-type cells ( Figure 1D).…”

“…Tung is a subtropical tree that produces a seed oil containing high amounts of eleostearic acid (18:3D 9cis,11trans,13trans ), an unusual conjugated fatty acid that imparts industrially useful drying qualities to the oil (Sonntag, 1979). We previously identified the enzyme required for eleostearic acid biosynthesis, a diverged, Cb5-dependent D 12 oleic acid desaturase (FAD2) termed FADX (Dyer et al, 2002), and subsequently determined the mechanisms for FAD targeting and assembly into ER membranes in plant cells (McCartney et al, 2004). Here, we report the identification, functional analysis, and a detailed description of the molecular targeting signals for four Cb5 proteins that are expressed in developing tung seeds during biosynthesis of tung oil.…”

Development of marker-free transgenic Jatrophaplants with increased levels of seed oleic acid

“…As shown in Additional file 2, the expression of FAD2-1 gene is high in seeds and weaker in vegetative tissues, while the FAD2-2 gene is expressed highly in seeds and not detectable in leaf tissue. The expression pattern of these two FAD2 genes in Jatropha is very similar to others in the Euphorbiaceae: FAD2 and Fatty acid hydroxylase 12 ( FAH12 ) in castor bean [13], FAD2 and FADX in the tung tree ( Aleurites fordii ) [12]. All the above data suggest that JcFAD2-2, similar to FADX and FAH12, may function more like an unusual fatty acid enzyme than a desaturase.…”