Enzymes of beta-Oxidation in Different Types of Algal Microbodies

The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H₂O₂-forming oxidase rather than a dehydrogenase.

The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae.

     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.     

Intracellular Localization of Enzymes of Fatty Acid-beta-Oxidation in the Alga Cyanidium caldarium

The intracellular distribution of enzymes, participating in the β-oxidation of fatty acids in the eucaryotic alga Cyanidium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and thiolase were present in the mitochondrial fraction (density: 1.19 gram per cubic centimeter). Activity of an acyl-CoA synthetase was found in the mitochondrial fraction as well as in a band where mitochondrial membrane apparently had accumulated (density: 1.17 gram per cubic centimeter). None of these enzymes were present in the peroxisomes (density: 1.23 gram per cubic centimeter). Results from cell fractionation as well as properties of β-oxidation enzymes indicate a mitochondrial location of fatty acid degradation also in the algae Galdieria sulphuraria and Cyanidioschyzon merolae.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Localisation of β-oxidation enzymes in peroxisomes of rice coleoptiles

Enzymes of the β-oxidation pathway in rice ( Oryza sativa L., cv. Arborio) coleoptiles were investigated. The coleoptiles contain acyl-CoA oxidase (EC 1.3.99.3), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), enoyl-CoA hydratase (EC 4.2.1.17) and thiolase (EC 2.3.1.9). Analysis of coleoptile homogenates by sucrose density fractionation showed a preferential distribution of these enzymes in the unspecialized peroxisomes. The enzymatic activity found in the mitochondrial fraction was due to peroxisomal contamination since electron micrographs show the peroxisomes to be intact and pure whereas the mitochondrial fraction was contaminated by other organelles. It appears that the β-oxidation pathway is localized in the unspecialized peroxisomes of rice coleoptiles, extending the number of plant species in which such a localization has been observed.     

Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat.

1. Changes in the activities of several enzymes involved in mitochondrial fatty acid oxidation were measured in livers of developing rats between late foetal life and maturity. The enzymes studied are medium- and long-chain ATP-dependent acyl-CoA synthetases of the outer mitochondrial membrane and matrix, GTP-dependent acyl-CoA synthetase, carnitine acyltransferase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase.     

Innermembrane association of three mitochondrial β-oxidation enzymes revealed by immunoelectron microscopic technique

Summary Ultrastructural localization of three mitochondrial β-oxidation enzymes, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase in rat liver was studied by a post-embedding immunocytochemical technique. Rat liver was fixed by perfusion. Vibratome sections (100 μm thick) were embedded in Lowicryl K4M. Ultrathin sections were separately incubated with antibody to each enzyme, followed by protein A-gold complex. Gold particles representing the antigenic sites for all enzymes examined were confined exclusively to mitochondria of hepatocytes and other sinus-lining cells. Peroxisomes were consistently negative for the immunolabelling. In the mitochondria the gold particles were localized in the matrical side of inner membrane. The control experiments confirmed the specificity of the immunolabelling. The results firstly indicate that the mitochondrial β-oxidation enzymes are present in the matrix of mitochondria and associated with the inner membrane.     

β-Oxidation of fatty acids in algae: Localization of thiolase and acyl-CoA oxidizing enzymes in three different organisms

In the algae Mougeotia, Bumilleriopsis and Eremosphaera, recently shown to possess the enzymes hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and enoyl-CoA hydratase (EC 4.2.1.17), the presence of thiolase (EC 2.3.1.9) and acyl-CoA-oxidizing enzymes can also be demonstrated, indicating that -oxidation of fatty acids is possible in these organisms. The compartmentation of enzymes is different in the various algae. In Mougeotia, both thiolase and the acyl-CoA-oxidizing enzyme are located exclusively in the peroxisomes. The latter enzyme was found to be an oxidase using molecular oxygen as an electron acceptor. On the other hand, in Bumilleriopsis all enzymes of the fatty-acid -oxidation pathway tested are constituents only of the mitochondria, and acyl-CoA is oxidized by a dehydrogenase incapable of reducing oxygen. Finally, in Eremosphaera thiolase and acyl-CoA-oxidizing enzymes were found in the peroxisomes as well as in the mitochondria. In the peroxisomes, oxidation of acyl-CoA is catalyzed by an oxidase, whereas the corresponding enzyme in the mitochondria is a dehydrogenase. The acyl-CoA oxidases/dehydrogenases of the three algae differ not only by their capability for oxidation of acyl-CoA of different chain lengths but also with regard to their K_m values and substrate specificities. Indications were obtained that the oxygen is reduced to water rather than to H₂O₂ by the algal acyl-CoA oxidases. When cells of Eremosphaera were cultured with hypolipodemic substances in the growth medium the activities of the peroxisomal enzymes, but not those of the mitochondrial enzymes of the fatty-acid -oxidation pathway, were increased by a factor of two to three.Abbreviations DPIP 2,6-dichlorophenol indophenol - INT p-iodonitrotetrazolium violet - MEHP monoethylhexylphthalate     

Innermembrane association of three mitochondrial beta-oxidation enzymes revealed by immunoelectron microscopic technique

Ultrastructural localization of three mitochondrial beta-oxidation enzymes, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase in rat liver was studied by a post-embedding immunocytochemical technique. Rat liver was fixed by perfusion. Vibratome sections (100 micron thick) were embedded in Lowicryl K4M. Ultrathin sections were separately incubated with antibody to each enzyme, followed by protein A-gold complex. Gold particles representing the antigenic sites for all enzymes examined were confined exclusively to mitochondria of hepatocytes and other sinus-lining cells. Peroxisomes were consistently negative for the immunolabelling. In the mitochondria the gold particles were localized in the matrical side of inner membrane. The control experiments confirmed the specificity of the immunolabelling. The results firstly indicate that the mitochondrial beta-oxidation enzymes are present in the matrix of mitochondria and associated with the inner membrane.     

Identification of enzymes involved in oxidation of phenylbutyrate

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.     

Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates.     

The large subunit of the pig heart mitochondrial membrane-bound β-oxidation complex is a long-chain enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

The subunit locations of the component enzymes of the pig heart trifunctional mitochondrial β-oxidation complex are suggested by analyzing the primary structure of the large subunit of this membrane-bound multienzyme complex [Yang S.-Y.et al. (1994) Biochem. biophys. Res. Commun. 198, 431–437] with those of the subunits of the E. coli fatty acid oxidation complex and the corresponding mitochondrial matrix β-oxidation enzymes. Long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are located in the amino-terminal and the central regions of the 79 kDa polypeptide, respectively, whereas the long-chain 3-ketoacyl-CoA thiolase is associated with the 46 kDa subunit of this complex. The pig heart mitochondrial bifunctional β-oxidation enzyme is more homologous to the large subunit of the prokaryotic fatty acid oxidation complex than to the peroxisomal trifunctional β-oxidation enzyme. The evolutionary trees of 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases suggest that the mitochondrial inner membrane-bound bifunctional β-oxidation enzyme and the corresponding matrix monofunctional β-oxidation enzymes are more remotely related to each other than to their corresponding prokaryotic enzymes, and that the genes of E. coli multifunctional fatty acid oxidation protein and pig heart mitochondrial bifunctional β-oxidation enzyme diverged after the appearance of eukaryotic cells.     

Partial purification and characterization of the mitochondrial and peroxisomal isozymes of enoyl-coenzyme a hydratase from germinating pea seedlings

Distinct organellar forms of the β-oxidation enzyme enoyl-coenzyme A (CoA) hydratase were partially purified and characterized from 2-day germinated pea (Pisum sativum L.) seedlings. The purification was accomplished by disruption of purified mitochondria or peroxisomes, (NH₄)₂SO₄ fractionation, and gel permeation chromatography using a column of Sephacryl S-300. The organellar isozymes had distinct kinetic constants for the substrates 2-butenoyl-CoA and 2-octenoyl-CoA, and could be easily distinguished by differences in thermostability and salt activation. The peroxisomal isozyme had a native M_r of 75,000 and appeared to be a typical bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, while the mitochondrial isozyme had a native M_r of 57,000 and did not have associated dehydrogenase activity. Western blots of total pea mitochondrial proteins gave a positive signal when probed with anti-rat liver mitochondrial enoyl-CoA hydratase antibodies but there was no signal when blots of total peroxisomal proteins were probed.     

Mitochondrial metabolism of valproic acid

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination : IV. beta-OXIDATION

Microbodies were isolated by zonal-rotor sucrose density gradient centrifugation from cotton (cv. DP 61) seeds at two distinct stages of embryogenesis (38 and 50 days after anthesis) and after 48 hours postgerminative growth. In all cases, β-oxidation activity (palmitoyl-coenzyme A (CoA)-dependent reduction of acetylpyridine adenine dinucleotide or production of acetyl-CoA) and activities of the enzymes palmitate:CoA ligase, acyl-CoA oxidase, enoyl hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, plus catalase, were localized exclusively in the microbody fractions, i.e. none of the activities were associated with mitochondria. Acyl-CoA dehydrogenase activity could not be detected in any of the gradient fractions or in homogenates.     

Induction of acyl coenzyme A synthetase and hydroxyacyl coenzyme A dehydrogenase during fatty acid degradation in Neurospora crassa

Neurospora crassa is able to use long-chain fatty acids as the sole carbon and energy source. After growth on oleate there was nearly a 10-fold induction of the acyl coenzyme A (CoA) synthetase and a fivefold increase in the activity of the 3-hydroxyacyl-CoA dehydrogenase. There was a slight induction of the enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase, but no apparent induction of the flavin-linked acyl-CoA dehydrogenase. These noncoordinate changes in the fatty acid degradation enzymes suggest that they are not organized into a multienzyme complex as is found in bacteria.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of beta-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and V(max.) and K(m) values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. V(max.), K(m) and K(i) values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the beta-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.     

Immunocytochemical demonstration of peroxisomal enzymes in human kidney biopsies

Peroxisomes are particularly abundant in the proximal tubules of the mammalian kidney. We describe the immunocytochemical localization of catalase and three peroxisomal lipid beta-oxidation enzymes: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase, in human renal biopsies fixed with glutaraldehyde and embedded in Epon. For light microscopy of semithin sections, satisfactory immunostaining required removal of the resin and controlled proteolytic digestion followed by the indirect immunoperoxidase technique. Brief etching of ultrathin sections with alkoxide followed by the protein A-gold method were used for electron microscopic localization of the enzymes. The immunoreactive peroxisomes were distinctly visualized in proximal tubular epithelial cells with no staining of any other cell organelles. The results establish the presence of catalase and of peroxisomal lipid beta-oxidation system proteins in human kidney. The immunocytochemical procedure described herein provides a simple approach for the investigation of peroxisomal structure and function in human renal biopsies processed for ultrastructural studies.     

Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

Polyunsaturated fatty acids promote the rapid fusion of lipid droplets in Caenorhabditis elegans

Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LD fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here, we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal β-oxidation pathway in Caenorhabditis elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show that dysfunction of peroxisomal β-oxidation results in the accumulation of long-chain fatty acid-CoA and phosphocholine, which may activate the sterol-binding protein 1/sterol regulatory element–binding protein to promote gLD formation. Furthermore, we found that inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids (PUFAs) with three or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or phosphocholine bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal β-oxidation–defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.