Identification of Non-Heme Diiron Proteins That Catalyze Triple Bond and Epoxy Group Formation

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Science  08 May 1998:
Vol. 280, Issue 5365, pp. 915-918
DOI: 10.1126/science.280.5365.915


Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Δ12 acetylenic bond in 9-octadecen-12-ynoic acid and the Δ12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non–heme iron that have histidine-rich motifs.

Over 600 naturally occurring acetylenic compounds are known, many of which occur in higher plants and mosses (1, 2). Knowledge of the enzymatic reactions leading to the formation of a carbon-carbon acetylenic (triple) bond is limited; it is known, however, that in the moss Ceratodon purpurea, an acetylenic bond at the Δ6 position in a C18 fatty acid is formed from a carbon-carbon double bond (3), whereas in Crepis rubra, oleate is a substrate in the synthesis of 9-octadecen-12-ynoic acid (crepenynic acid) (4).

We have studied the synthesis of acetylenic and epoxy fatty acids in plants of the genus Crepis (2, 5). Crepis alpina seed oil is made up of about 70% crepenynic acid, andCrepis palaestina seed oil is made up of about 60% vernolic acid (12,13-epoxy-9-octadecenoic acid). Here we characterized the enzymes involved in the biosynthesis of crepenynic and vernolic acid inC. alpina and C. palaestina (6). Microsomes from developing seeds of C. alpina, prepared in the presence of reduced nicotinamide adenine dinucleotide (NADH), converted [14C]linoleate into [14C]crepenynate (Table 1). Similarly, microsomes from developing seeds of C. palaestinaconverted [14C]linoleate into [14C]vernoleate. The results indicated that both the Δ12 acetylenic bond–forming and Δ12 epoxidation reactions used acyl chains with a carbon double bond at the Δ12 position as substrate.

Table 1

Properties of different fatty acid modification enzymes [some of these results were published earlier (6,7)]. The amount of enzyme activity is given as the amount of radioactive substrate converted into product relative to a control, which was in each case the incubation conditions giving the highest activity. The amount of substrate conversion in these controls was C. alpina acetylenase, 12%; C. palaestinaepoxygenase, 26%; C. alpina desaturase, 30%; and E. lagascae epoxygenase, 13%.

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These reactions, and the Δ12 desaturase, required NADH or NADPH and were inhibited by cyanide (Table 1). Unlike the Euphorbia lagascae epoxygenase, which is likely to be a cytochrome P-450–type enzyme (7), the C. palaestinaepoxygenase was unaffected by carbon monoxide or antibodies to cytochrome P-450 reductase (8). Both the Δ12 epoxygenation and Δ12 acetylenation enzymes of Crepis had biochemical characteristics typical of fatty acid desaturases and might therefore, we thought, be structurally similar to these enzymes (9,10).

To clone the gene encoding the acetylenase, we used cDNA from C. alpina developing seed and primers derived from endoplasmic reticular (ER) desaturases (11) for polymerase chain reaction (PCR). DNA sequencing of the products revealed a likely ER Δ15 desaturase sequence, a likely Δ12 desaturase sequence (D12N), and a variant ER Δ12 sequence (D12V). Northern (RNA) blot analysis showed that expression of the D12V sequence was seed specific inC. alpina, as was the presence of crepenynic acid.

The D12V fragment was used to isolate a full-length cDNA (Crep1) from aC. alpina developing-seed library from which the plasmid pCrep1 was produced (12). Sequencing of Crep1 revealed a putative protein of 375 amino acids (Fig.1). This protein had 59% identity to the castor bean Δ12-hydroxylase and 56% identity to theArabidopsis ER Δ12 desaturase when compared pairwise.

Figure 1

Alignment of derived amino acid sequences. The amino acid sequences of the C. palaestina epoxygenase (epox),C. alpina acetylenase (acet), C. palaestinaputative Δ12 desaturase (cpde), A. thaliana Δ12 desaturase (atde), and castor bean oleate hydroxylase (hydr) (30) were aligned and from this a consensus sequence (cons) generated with the programs Pileup and Pretty (31). The consensus sequence is in uppercase and variant residues are in lowercase. Dots represent gaps introduced to optimize alignment, and hyphens indicate residues that are identical to the analogous amino acid in the consensus sequence. The three histidine-rich motifs [HX(3 or 4)H, HX(2 or 3)HH, HX(2 or 3)HH] (22) are underlined. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

The Crep1 gene was expressed in Saccharomyces cerevisiae(YN94-1 strain) with the plasmid pVT-Crep1 (13). Yeast were cultivated in media with linoleic acid and their fatty acid composition determined (14). A peak with the same retention time as the methyl ester of crepenynic acid (up to 0.3% of the total peak area) was identified in the pVT-Crep1–transformed yeast but not in yeast transformed with vector alone. In addition, only the pVT-Crep1–transformed yeast had fatty acid diethylamides (FADEAs), prepared from the total fatty acids and analyzed by gas-liquid chromatography–mass spectrometry (GLC-MS) (15), that contained a compound with the same retention time and mass of 9-octadecen-12-ynoic acid diethylamide (Fig.2, A and B). The mass spectrum of this FADEA was identical to the FADEA derivative of crepenynic acid (Fig. 2C). Thus, the Crep1 gene encodes an enzyme that catalyzes the formation of an acetylenic bond from the Δ12 double bond of linoleate to give crepenynic acid. We designated this type of enzyme acetylenase, because it is capable of converting a carbon-carbon double bond into an acetylenic bond and is distinct from the related desaturases that convert carbon single bonds into double bonds.

Figure 2

GLC-MS analysis of fatty acid derivatives from transgenic yeast. (A) Single-ion chromatograms [367 atomic mass units (amu) and 333 amu] of FADEA derivatives from yeast transformed with pVT-Crep1 and cultivated with linoleic acid. The peaks indicated correspond to mass ions of eicosanoic acid (367 amu, peak 1) and crepenynic acid (333 amu, peak 2). (B) Single-ion chromatograms of FADEA derivatives from yeast with vector alone and cultivated with linoleic acid. Peak 1 is the mass ion of eicosanoic acid (367 amu). (C) Mass spectrum of the compound giving rise to peak 2 in chromatogram (A). m/z, mass-to-charge ratio.

Yeast containing pVT-Crep1 and grown without linoleic acid could produce linoleic acid (up to 0.4% w/w of total fatty acids) in contrast to the control yeast. Although the C. alpinaacetylenase has Δ12 desaturase activity, crepenynic acid was not detected in these yeast, implying that the linoleate produced was not used to synthesize crepenynic acid. The D12N sequence, also expressed in developing C. alpina seeds, probably represents the ER Δ12 desaturase that produces the linoleate used by the acetylenase.

We expressed the acetylenase gene in Arabidopsis with the seed-specific napin promoter (16). Total fatty acids from seeds of individual T0 transgenic plants contained up to 25% (w/w) crepenynic acid in contrast to control plants (Fig.3). No other acetylenic fatty acids were detected (17).

Figure 3

GLC analysis of A. thaliana seed fatty acids. Methyl esters of seeds from a plant transformed with (A) empty vector, (B) the C. alpinaacetylenase, and (C) the C. palaestinaepoxygenase were analyzed and their fatty acid profiles determined. In (A) the indicated peaks correspond to methyl ester (Me) derivatives of hexadecanoic acid, 1; octadecanoic acid, 2; 9-octadecenoic acid, 3; 9,12-octadecadienoic acid, 4; 9,12,15-octadecatrienoic acid, 5; 11-eicosaenoic acid, 6; and 13-docosaenoic acid, 7. In (B) and (C) the peaks corresponding to the methyl ester derivatives of crepenynic and vernolic acid are indicated.

To clone the Δ12 epoxygenase gene, we used the D12V fragment to screen a cDNA library from C. palaestina developing seeds (18). A clone (Cpal2) was isolated that encoded a putative protein of 374 amino acids. This amino acid sequence was similar to theC. alpina acetylenase (81% identity), theArabidopsis ER Δ12-desaturase (58% identity), and the castor bean Δ12-hydroxylase (53% identity) when compared pairwise. A putative Δ12 desaturase clone was also isolated from this C. palaestina library (Fig. 1).

We transformed the Cpal2 cDNA into Arabidopsis(19). Total fatty acids from seeds of T0transgenic plants contained up to 15% (w/w) of vernolic acid (Fig. 3). Seeds from some plants also had up to 1% (w/w) of an epoxy fatty acid identified tentatively as 12-epoxy-9,15-octadecadienoic acid (20). This fatty acid probably arose through Δ15 desaturation of vernolic acid by the endogenous Arabidopsisenzyme because developing linseeds can desaturate added vernolic acid at the Δ15 position (21).

Despite the homology between the Crep1 and Cpal2 sequences, acetylenic and epoxy fatty acids were not detected in seeds fromArabidopsis plants carrying the epoxygenase or acetylenase gene, respectively. It appears that only moderate changes in amino acid sequence may determine whether these enzymes are acetylenases or epoxygenases. Their shared sequence differences to the Δ12 desaturases might reflect the changes needed for the recognition of a linoleate instead of an oleate substrate. Sequence comparison of the oleate hydroxylase and the expression of a mutant enzyme suggested that as few as six amino acids might determine if this enzyme is a desaturase or hydroxylase (22). That both crepenynic and vernolic acid can occur in the seed of some Crepis species (2) may indicate that these species contain both an acetylenase and an epoxygenase or a dual-functional enzyme.

The sequence and biochemical characteristics of the acetylenase and epoxygenase suggest that they are likely to be non–heme diiron proteins (Fig. 1) (23, 24). This group of proteins includes desaturases, hydroxylases, and epoxygenases found in animals, fungi, plants, and bacteria (9, 25-27). The diverse reactions that these enzymes catalyze probably use a common reactive center (10). Histidine-rich motifs are thought to form part of the diiron center where oxygen activation and substrate oxidation occur (26, 28). At least four reactions (Fig.4) can be catalyzed by Δ12 desaturase–like plant enzymes.

Figure 4

Reactions catalyzed by plant Δ12 desaturase and Δ12 desaturase-like enzymes. Reactions A, B, C, and D can be catalyzed by a Δ12 desaturase, Δ12 hydroxylase, Δ12 acetylenase, and Δ12 epoxygenase, respectively.

Many of the unusual plant fatty acids are potentially valuable for the production of paints, varnishes, plastisizers, resins, lubricants, and polymers. Crepenynic acid, for example, can be converted by alkali isomerization to 8,10,12-octadecatrienoic acid and C18cyclohexadiene monocarboxylic acid (29), which are valuable in high-quality coatings and cold weather ester-type lubricants, respectively. Similarly, epoxidized fatty acids are widely used as plastisizers. The production of large amounts of epoxy and acetylenic fatty acid in transgenic seeds may contribute to converting traditional agricultural crops into efficient producers of more valuable chemical commodities. Commercial production of such crops might be a reality in the near future.

  • * To whom correspondence should be addressed. E-mail: sten.stymne{at}


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