Glyoxysomal β-oxidation of long-chain fatty acids: completeness of degradation

The ability of glyoxysomes from sunflower (Helianthusannuus L.) cotyledons to completely degrade long-chain fatty acids into their constituent acetyl units and the time courses of the appearance of acyl-CoA intermediates during β-oxidation have been studied using ¹⁴C-labelled substrates at non-saturating concentrations (1.3 to 1.8 μmol · l⁻¹). [¹⁴C]Acetyl-CoA was formed from [18-¹⁴C]oleate metabolized at a yield of up to 80%, and from [U-¹⁴C]palmitate and [U-¹⁴C]linoleate to an extent indicating that a maximum of 80% and 30%, respectively, of the substrate β-oxidized had been degraded beyond the C₄-CoA intermediate level. To obtain the latter values, an acetyl-CoA-removing system was required during β-oxidation. Constant re-oxidation of the NADH formed during the β-oxidation did not replace the effect of acetyl-CoA removal. Neither the completeness of the linoleate β-oxidation nor the rate of reaction were influenced by NADPH. Medium- and short-chain acyl-CoA intermediates were predominantly detected during β-oxidation of the long-chain substrates employed. The degradation of these intermediates appeared to be stimulated mainly in the presence of an acetyl-CoA-removing system. The time courses of the appearance of intermediates corresponded to a precursor-product relationship between intermediates of decreasing chain lengths. Received: 12 December 1997 / Accepted: 26 January 1998     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

β-Oxidation of 2-ethyl-5-carboxypentyl phthalate in rodent liver

[7-¹⁴C]2-Ethyl-5-carboxypentyl phthalate was isolated and purified from urine of rats given [7-¹⁴C]-di-(2-ethylhexyl) phthalate. This metabolite was shown to serve as a precursor for 2-ethyl-3-carboxypropylphthalate in vivo. 2-Ethyl-5-carboxypentyl phthalate was oxidized to 2-ethyl-3-carboxypropyl phthalate in liver slices from control or, much more rapidly, from clofibrate-pretreated rats. Inhibition by KCN in liver slices from untreated rats, and strong inhibition by acrylate, suggested that formation of 2-ethyl-3-carboxy-propyl phthalate involved mitochondria β-oxidation. The strong enhancement of the product of this compound by clofibrate (a very weak inducer for mitochondrial dehydrogenases), and strong inhibition by chlorpromazine suggested that peroxisomes may also be able to oxidize 2-ethyl-5-carboxypentyl phthalate. We were able to detect β-oxidation of 2-ethyl-5-carboxypentyl phthalate to 2-ethyl-3-carboxypropyl phthalate using purified mitochondria, but strong phthalate monoester hydrolase activity observed during incubation of the former compound with purified peroxisomes made it impossible to determine whether 2-ethyl-3-carboxypropyl phthalate could be produced in the latter organelle or not. 2-Ethyl-5-carboxypentyl phthalate was such an inefficient substrate for β-oxidation compared to palmitic acid that it is unlikely that it contributes significantly to the production of H₂O₂ in rats chronically exposed to di-(2-ethylhexyl) phthalate. Normal fatty acids are most likely to serve as the dominat substrates for peroxisomal β-oxidase.     

Acetylaspartate as an extramitochondrial physiological carrier of acetyl coA for fatty acid synthesis

Aminooxyacetate, a transaminase inhibitor, suppresses the enrichment in radioactivity found in the fatty acids of animals receiving 2, 4-¹⁴C citrate in comparison with 1, 5-¹⁴C citrate. On the other hand 3H from N-acetyl-³H aspartate is significantly incorporated into fatty acids $\text{in vivo}$ or in presence of liver supernatant fractions. Our results indicate that citrate seems to be an effective carrier of acetyl CoA for fatty acid synthesis mainly in the rat liver and that acetylaspartate may be an other physiological carrier of acetyl CoA outside the mitochondria.     

Peroxisomal lipid degradation via β-and α-oxidation in Mammals

Peroxisomal β-oxidation is involved in the degradation of long chain and very long chain fatty acyl-(coenzyme A)CoAs, long chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs (e.g. pristanoyl-CoA), and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (side chain of cholesterol). In the rat, straight chain acyl-CoAs (including the CoA esters of dicarboxylic fatty acids and eicosanoids) are β-oxidized via palmitoyl-CoA oxidase, multifunctional protein-1 (which displays 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA, dehydrogenase activities) and peroxisomal thiolase. 2-Methyl-branched acyl-CoAs are degraded via pristanoyl-CoA oxidase, multifunctional protein-2 (MFP-2) (which displays 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activities) and sterol carrier protein-X (SCPX; displaying 2-methyl-3-oxoacyl-CoA thiolase activity). The side chain of the bile acid intermediates is shortened via one cycle of β-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase, MFP-2 and SCPX. In the human, straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase, multifunctional protein-1, and peroxisomal thiolase, as is the case in the rat. The CoA esters of 2-methyl-branched acyl-CoAs and the bile acid intermediates, which also possess a 2-methyl substitution in their side chain, are shortened, via branched chain acyl-CoA oxidase (which is the human homolog of trihydroxycoprostanoyl-CoA oxidase), multifunctional protein-2, and SCPX. The rat and the human enzymes have been purified, cloned, and kinetically and stereochemically characterized. 3-Methyl-branched fatty acids such as phytanic acid are not directly β-oxidizable because of the position of the methyl-branch. They are first shortened by one carbon atom through the a-oxidation process to a 2-methyl-branched fatty acid (pristanic acid in the case of phytanic acid), which is then degraded via peroxisomal β-oxidation. In the human and the rat, α-oxidation is catalyzed by an acyl-CoA synthetase (producing a 3-methylacyl-CoA), a 3-methylacyl-CoA 2-hydroxylase (resulting in a 2-hydroxy-3-methylacyl-CoA), and a 2-hydroxy-3-methylacyl-CoA lyase that cleaves the 2-hydroxy-3-methylacyl-CoA into a 2-methyl-branched fatty aldehyde and formyl-CoA. The fatty aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl-branched fatty acid while formyl-CoA is hydrolyzed to formate, which is then converted to CO₂. The activation, hydroxylation and cleavage reactions and the hydrolysis of formyl-CoA are performed by peroxisomal enzymes; the aldehyde dehydrogenation remains to be localized whereas the conversion of formate to CO₂ occurs mainly in the cytosol.     

Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Fatty acid cellular metabolism and lactone production by the yeast Pichia guilliermondii

Methyl ricinoleate conversion into γ-decalactone by fungi is already widely used by the aromatic industry. It offers an interesting alternative to chemical synthesis by permitting acquisition of a natural label. Peroxisomal β-oxidation has been described as the probable transformation mechanism. This paper provides information about this metabolism and shows the importance of the step catalysed by carnitine octanoyltransferase. After culture of the yeast Pichia guilliermondii on a medium containing methyl ricinoleate as sole carbon source, we confirmed that mitochondrial β-oxidation could not be responsible for the biotransformation. We also observed the effect of chlorpromazine, an inhibitor of carnitine octanoyltransferase, on peroxisomal β-oxidation and therefore on lactone production, and on lipid accumulation by the yeasts. The presence of chlorpromazine caused a reduction in aromatic specific production yield. This reduction was inversely proportional to the amount of chlorpromazine present in the medium. A considerable accumulation of methyl ricinoleate derivatives was also observed. We therefore concluded that the metabolism responsible for the bioconversion was peroxisomal β-oxidation. The effects of chlorpromazine suggested that the entry of fatty acids into the peroxisomes took place in a carnitine-dependent manner. This step might be a limiting step in the metabolism. Received: 26 June 1995/Received revision: 16 November 1995/Accepted: 4 December 1995     

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO₂ by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the β-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall β-oxidation activity was assayed as the formation of [¹⁴C]acetyl-CoA from [¹⁴C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the β-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

PNU-91325 increases fatty acid synthesis from glucose and mitochondrial long chain fatty acid degradation: a comparative tracer-based metabolomics study with rosiglitazone and pioglitazone in HepG2 cells

The mitochondrial membrane protein termed “mitoNEET,” is a putative secondary target for insulin-sensitizing thiazolidinedione (TZD) compounds but its role in regulating metabolic flux is not known. PNU-91325 is a thiazolidinedione derivative which exhibits high binding affinity to mitoNEET and lowers cholesterol, fatty acid and blood glucose levels in animal models. In this study we report the stable isotope-based dynamic metabolic profiles (SIDMAP) of rosiglitazone, pioglitazone and PNU-91325 in a dose-matching, dose-escalating study. One and 10 μM concentrations 1 and 10 μM drug concentrations were introduced into HepG2 cells in the presence of either [1,2−¹³C₂]-D-glucose or [U−¹³C₁₈]stearate, GC/MS used to determine positional tracer incorporation (mass isotopomer analysis) into multiple metabolites produced by the Krebs and pentose cycles, de novo fatty acid synthesis, long chain fatty acid oxidation, chain shortening and elongation. Rosiglitazone and pioglitazone (10 μM) increased pentose synthesis from [U−¹³C₁₈]stearate by 127% and 185%, respectively, while PNU-91325 rather increased glutamate synthesis in the Krebs cycle by 113% as compared to control vehicle treated cells. PNU-91325 also increased stearate chain shortening into palmitate by 59%. Glucose tracer-derived de novo palmitate and stearate synthesis were increased by 1 and 10 μM rosiglitazone by 41% and 83%, respectively, and by 63% and 75% by PNU-91325. Stearate uptake was also increased by 10 μM PNU-91325 by 15.8%. We conclude that the entry of acetyl Co-A derived from long-chain fatty acid β-oxidation into the mitochondria is facilitated by the mitoNEET ligand PNU-91325, which increases glucose-derived long chain fatty acid synthesis and breakdown via β-oxidation and anaplerosis in the mitochondria.     

Effect of clofibrate on the CoA thioester profile in rat liver.

Acute and chronic treatment with clofibrate increased the total CoA content of rat liver and altered the profile of the various CoA thioesters. There resulted a 2–3 fold increase in the contents of long chain acyl CoA, acetyl CoA and free CoA, contrasting with significant decreases found in succinyl CoA, malonyl CoA and acetoacetyl CoA contents. It is postulated that the known increase in fatty acid binding protein and/or the increased extramitochondrial β-oxidation of fat by an increased peroxisomal population may direct the compartmentation and metabolic fate of fatty acids and their CoA derivatives following clofibrate treatment.     

Sulfides impair short chain fatty acid β-oxidation at acyl-CoA dehydrogenase level in colonocytes: Implications for ulcerative colitis

The disease process of ulcerative colitis (UC) is associated with a block in -oxidation of short chain fatty acid in colonic epithelial cells which can be reproduced by exposure of cells to sulfides. The aim of the current work was to assess the level in the -oxidation pathway at which sulfides might be inhibitory in human colonocytes. Isolated human colonocytes from cases without colitis (n = 12) were exposed to sulfide (1.5 mM) in the presence or absence of exogenous CoA and ATP. Short chain acyl-CoA esters were measured by a high performance liquid chromatographic assay. ¹⁴CO₂ generation was measured from [1-¹⁴C]butyrate and [6-¹⁴C]glucose. ¹⁴CO₂ from butyrate was significantly reduced (p < 0.001) by sulfide. When colonocytes were incubated with hydrogen sulfide in the presence of CoA and ATP, butyryl-CoA concentration was increased (p < 0.01), while crotonyl-CoA (p < 0.01) and acetyl-CoA (p < 0.01) concentrations were decreased. These results show that sulfides inhibit short chain acyl-CoA dehydrogenase. As oxidation of n-butyrate governs the epithelial barrier function of colonocytes the functional activity of short chain acyl-CoA dehydrogenase may be critical in maintaining colonic mucosal integrity. Maintaining the functional activity of dehydrogenases could be an important determinant in the expression of ulcerative colitis.     

Incorporation of acetyl-CoA generated from peroxisomal β-oxidation into ethanolamine plasmalogen of rat liver

We have reported that peroxisomal β-oxidation has an anabolic function, supplying acetyl-CoA for biosynthesis of bile acids and phospholipids. Here we deal with its role in the biosynthesis of the subclasses of ethanolamine- and choline-containing phosphoglycerides (EPG, CPG, respectively). Rats were fed for 2 weeks on chow containing 0.25% clofibrate, which inhibits cholesterol and bile acid biosyntheses, but stimulates peroxisomal β-oxidation. [1-¹⁴C]Lignoceric acid, which is exclusively degraded by peroxisomal β-oxidation to acetyl-CoA, was intravenously injected, and 3 h later the rats were killed. The EPG-rich and CPG-rich fractions were prepared from the liver. When they were treated with phospholipase A₂, the radioactivity was predominantly recovered in the 1-radyl group. The radioactivity in EPG was easily dissociated with HCl vapor, and the lipid containing radioactivity was found to be a fatty aldehyde mixture consisting of steary aldehyde (approx. 58%) palmityl aldehyde (approx. 40%) and oleyl aldehyde (approx. 2%). Thus, in the case of EPG, acetyl-CoA from peroxisomal β-oxidation is incorporated mainly into the 1-alkenyl group of ethanolamine plasmalogen. The radioactivity in CPG, however, was found in fatty alcohol (formed from fatty acid), but not in alkylglycerol after reduction of the fraction with Vitride. Thus, in the case of CPG, acetyl-CoA from peroxisomal β-oxidation is exclusively incorporated into the 1-acyl group of diacyl glycerophosphocholine, but not into the 1-alkyl group. The above results were supported by the results of phospholipase C treatment. The above data indicate that peroxisomal β-oxidation plays a role in supplying acetyl-CoA for 1-alkenyl group of plasmalogen-type phospholipid, but this channel may open only to synthesis of EPG, and almost not to CPG.     

Modulation of peroxisomal and microsomal fatty acid oxidation by acetone. A comparative study between liver and kidney

The effect of acetone consumption on some microsomal and peroxisomal activities was studied in rat kidney and these results were compared with data from former investigations in liver. Acetone increased the microsomal lauric acid hydroxylation, the aminopyrine N-demethylation catalyzed by cytochrome P450 and the microsomal UDP-glucuronyltransferase activity. Also, acetone increased the peroxisomal β-oxidation of palmitoyl CoA and catalase activities in kidney. These studies suggest that acetone is a common inducer of the microsomal and peroxisomal fatty acid oxidation, as previously shown in both starved and ethanol treated rats. Our results support the hypothesis that microsomal fatty acid ω-hydroxylation results in the generation of substrates being supplied for peroxisomal β-oxidation. We propose that the final purpose of these linked fatty acid oxidations could be the catabolism of fatty acids or the generation of a substrate for the synthesis of glucose from fatty acids. This pathway would be triggered by acetone treatment in a similar way in liver and kidney.     

Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on β-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall β-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall β-oxidation activity but reduced the γ-decalactone production rates. This seemed to indicate a non-rate-limiting role for β-oxidation in the biotransformation of ricinoleic acid to γ-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed γ-decalactone. We checked the ability of the different strains to consume γ-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We␣concluded that peroxisomal β-oxidation is certainly involved in γ-decalactone catabolism by the yeast Y.␣lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. Received: 13 June 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Auxin-induced growth and its linkage to potassium channels

This study addresses the still open question of whether or not in oily storage tissues, e.g. cotyledons of germinating rape (Brassica napus L.) seedlings' lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and the β-oxi-dation system of fatty acids are located in one or more membrane-bounded organelles. The organelles were isolated carefully and identified by marker-enzyme activities. Activities of neither lipase nor acylester acylhydrolase (EC 3.1.1) could be detected either in glyoxysomes or in mitochondria, even when various substrate emulsions were employed. Only after long-term incubations could the presence of a low lipolytic activity be demonstrated for different organellar fractions. This alkaline carboxylic ester hydrolase, whose activity is below the detection limit of various standard tests, cannot play a role in the lipolytic function of glyoxysomes. In addition, a complete set of enzyme activities necessary for the conversion of saturated fatty acids to acetyl CoA was found only in the glyoxysomal cell fraction. The low β-oxidation activity discovered in the mitochondrial cell fraction is evidently due to glyoxysomal contamination. Enzyme activities unique to the mitochondrial β-oxidation system such as carnitine palmitoyltransferase (EC 2.3.1.21), carnitine acetyltransferase (EC 2.3.1.7), and acyl-CoA dehydrogenase (EC 1.3.99.3) were absent, indicating that mitochondria are not involved in fatty acid metabolism. In addition, on Western blots, antibodies raised against malate synthase (EC 4.1.3.2) and acyl-CoA oxidase (EC 1.1.3) recognized three polypeptides with molecular masses of 45, 63, and 70 kDa only in glyoxysomal fractions. Obviously, in the fatty rape seed neither glyoxysomes nor mitochondria are involved in triacylglycerol hydrolysis, and β-oxidation of fatty acids occurs exclusively in glyoxysomes. Received: 24 June 1996 / Accepted: 29 November 1996     

Total and peroxisomal fatty acid oxidation by liver homogenates from autopsied Reye's and control subjects

To determine whether the accumulation of liver triglyceride in Reye's syndrome could be due to a block in beta-oxidation of the fatty acids, the ability of Reye's and control liver homogenates from samples obtained at autopsy to oxidize fatty acids was examined. Total fatty acid oxidation as measured by oxidation of [1-14C]oleoyl CoA, which mostly represents mitochondrial activity, was comparable between the groups. Peroxisomal fatty acid oxidation was, likewise, similar despite the reported increase in the numbers and sizes of these organelles. This disparity could not be explained by an artifactual dilution of product by accumulated endogenous substrate. Inference is made that active peroxisomal beta-oxidation may contribute to the increased short chain fatty CoA content of liver which was reported earlier.     

IBR3, a novel peroxisomal acyl-CoA dehydrogenase-like protein required for indole-3-butyric acid response

Indole-3-butyric acid (IBA) is an endogenous auxin that acts in Arabidopsis primarily via its conversion to the principal auxin indole-3-acetic acid (IAA). Genetic and biochemical evidence indicates that this conversion is similar to peroxisomal fatty acid β-oxidation, but the specific enzymes catalyzing IBA β-oxidation have not been identified. We identified an IBA-response mutant (ibr3) with decreased responses to the inhibitory effects of IBA on root elongation or the stimulatory effects of IBA on lateral root formation. However, ibr3 mutants respond normally to other forms of auxin, including IAA. The mutant seedlings germinate and develop normally, even in the absence of sucrose, suggesting that fatty acid β-oxidation is unaffected. Additionally, double mutants between ibr3 and acx3, which is defective in an acyl-CoA oxidase acting in fatty acid β-oxidation, have enhanced IBA resistance, consistent with a distinct role for IBR3. Positional cloning revealed that IBR3 encodes a putative acyl-CoA dehydrogenase with a consensus peroxisomal targeting signal. Based on the singular defect of this mutant in responding to IBA, we propose that IBR3 may act directly in the oxidation of IBA to IAA. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H₂O₂-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of ¹⁴C-labeled acid-soluble products from 1-¹⁴C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H₂O₂ production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.     

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial β-oxidation pathways. Prior to channelling the fatty acids into β-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB). FaaB was found to be the major long-chain synthetase for fatty acid degradation. FaaB was shown to localise to the peroxisomes, and the corresponding gene was induced in the presence of short and long chain fatty acids. Deletion of the faaB gene leads to a reduced/abolished growth on a variety of fatty acids. However, at least one additional fatty acyl-CoA synthetase with a preference for short chain fatty acids and a potential mitochondrial candidate (AN4659.3) has been identified via genome analysis.