Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans

The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5' region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae.     

Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Isolation of rat liver peroxisomes by vertical-rotor centrifugation by using a self-generated, iso-osmotic, Percoll gradient.

1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog

Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode respectively an oxidoreductase and methyltransferase. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B(1) (AFB(1)). Aspergillus nidulans harbors a gene cluster that produces ST, as the aflQ and aflP orthologs are either non-functional or absent in the genome. Aspergillus ochraceoroseus produces both AF and ST, and it harbors an AF/ST biosynthetic gene cluster that is organized much like the A. nidulans ST cluster. The A. ochraceoroseus cluster also does not contain aflQ or aflP orthologs. However the ability of A. ochraceoroseus to produce AF would indicate that functional aflQ and aflP orthologs are present within the genome. Utilizing degenerate primers based on conserved regions of the A. flavus aflQ gene and an A. nidulans gene demonstrating the highest level of homology to aflQ, a putative aflQ ortholog was PCR amplified from A. ochraceoroseus genomic DNA. The A. ochraceoroseus aflQ ortholog demonstrated 57% amino acid identity to A. flavus AflQ. Transformation of an O-methylsterigmatocystin (OMST)-accumulating A. parasiticus aflQ mutant with the putative A. ochraceoroseus aflQ gene restored AF production. Although the aflQ gene does not reside in the AF/ST cluster it appears to be regulated in a manner similar to other A. ochraceoroseus AF/ST cluster genes. Phylogenetic analysis of AflQ and AflQ-like proteins from a number of ST- and AF-producing Aspergilli indicates that A. ochraceoroseus might be ancestral to A. nidulans and A. flavus.     

Effect of ischemia and reperfusion of the myocardium on in vitro beta-oxidation of fatty acids

The in vivo oxidation of perfused [14C]-labeled fatty acids has been shown to decrease dramatically in hypoxic hearts. This study addresses the influence of ischemia and reperfusion on the enzymic activities of beta-oxidation of fatty acids in mitochondria and of peroxisomal origin. The rate of beta-oxidation of fatty acids in the isolated mitochondria from myocardium of swine fed control diet declined about 20% by the ischemic insult induced by hypothermic cardioplegic arrest. Upon reperfusion, the rate of mitochondrial beta-oxidation returned to a normal level. In clofibrate-fed animals, the rate of mitochondrial beta-oxidation did not vary significantly between control, ischemic, and perfused tissues. Furthermore, neither in control nor in clofibrate-fed animals did the rates of peroxisomal beta-oxidation of fatty acids vary significantly in the ischemic or reperfused tissues as compared to that of preischemic controls. These results suggest that ischemia does not contribute to any loss of enzymic activity in beta-oxidation of fatty acid cycles either in mitochondria or peroxisomes. Furthermore, the feeding of 0.5% (w/w) clofibrate to pigs increased the rate of mitochondrial beta-oxidation of fatty acids only by 50% while that of peroxisomes increased threefold. A similar threefold increase in catalase activity was also produced by clofibrate feeding. These results suggest that the heart plays a role in the hypolipidemic action of clofibrate.     

Disruption of mitochondrial beta -oxidation of unsaturated fatty acids in the 3,2-trans-enoyl-CoA isomerase-deficient mouse

Cellular energy metabolism is largely sustained by mitochondrial beta-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci(-/-) mouse). ECI is the link in mitochondrial beta-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial beta-oxidation of unsaturated fatty acids is interrupted in eci(-/-)mice at the level of their respective 3-cis- or 3-trans-enoyl-CoA intermediates. Fasting eci(-/-) mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci(-/-) mice together with peroxisomal beta- and microsomal omega-oxidation enzymes. Combined peroxisomal beta- and microsomal omega-oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci(-/-) mouse might be a model of a yet undefined inborn mitochondrial beta-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the beta-oxidation spiral of saturated fatty acids.     

MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO(2). Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role of this enzyme in mammals is unknown. A computer-based search for novel peroxisomal proteins led to the identification of a candidate gene for human MCD, which encodes a protein with a canonical type-1 peroxisomal targeting signal of serine-lysine-leucine(COOH). We observed that recombinant MCD protein has high intrinsic malonyl-CoA decarboxylase activity and that a malonyl-CoA decarboxylase-deficient patient has a severe mutation in the MCD gene (c.947-948delTT), confirming that this gene encodes human MCD. Subcellular fractionation experiments revealed that MCD resides in both the cytoplasm and peroxisomes. Cytoplasmic MCD is positioned to play a role in the regulation of cytoplasmic malonyl-CoA abundance and, thus, of mitochondrial fatty acid uptake and oxidation. This hypothesis is supported by the fact that malonyl-CoA decarboxylase-deficient patients display a number of phenotypes that are reminiscent of mitochondrial fatty acid oxidation disorders. Additional support for this hypothesis comes from our observation that MCD mRNA is most abundant in cardiac and skeletal muscles, tissues in which cytoplasmic malonyl-CoA is a potent inhibitor of mitochondrial fatty acid oxidation and which derive significant amounts of energy from fatty acid oxidation. As for the role of peroxisomal MCD, we propose that this enzyme may be involved in degrading intraperoxisomal malonyl-CoA, which is generated by the peroxisomal beta-oxidation of odd chain-length dicarboxylic fatty acids.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

Subcellular localization and physiological role of alpha-methylacyl-CoA racemase

alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (S)-stereoisomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. Patients with a deficiency of alpha-methylacyl-CoA racemase accumulate in their plasma pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid, which are all substrates of the peroxisomal beta-oxidation system. Subcellular fractionation experiments, however, revealed that both in humans and rats alpha-methylacyl-CoA racemase is bimodally distributed to both the peroxisome and the mitochondrion. Our findings show that the peroxisomal and mitochondrial enzymes are produced from the same gene and that, as a consequence, the bimodal distribution pattern must be the result of differential targeting of the same gene product. In addition, we investigated the physiological role of the enzyme in the mitochondrion. Both in vitro studies with purified heterologously expressed protein and in vivo studies in fibroblasts of patients with an alpha-methylacyl-CoA racemase deficiency revealed that the mitochondrial enzyme plays a crucial role in the mitochondrial beta-oxidation of the breakdown products of pristanic acid byconverting (2R,6)-dimethylheptanoyl-CoA to its (S)-stereoisomer.     

Peroxisomal beta-oxidation: insights from comparative biochemistry

The activities of fatty acyl-CoA oxidase (FAO) and carnitine palmitoyl transferase (CPT), indices of the capacities of peroxisomal beta-oxidation and mitochondrial beta-oxidation, respectively, were determined in livers of several vertebrate species notable for differences in dietary fatty acid composition. In suckling rats FAO activities were half that in adult rats and CPT/FAO ratios twice that of adult rats. As their milk diet is dominated by medium chain fatty acids, this observation is consistent with current ideas about the role of peroxisomal beta-oxidation in rat liver in oxidation of long chain unsaturated fatty acids. In nectar-feeding hummingbirds (fatty acids synthesized de novo) FAO activities were 50% greater than adult rats and CPT/FAO ratios one-third less than adult rats, suggesting that peroxisomal beta-oxidation is relatively more important in this species, despite a fatty-acid-poor diet. In marine fish (herring, dogfish shark, hagfish) FAO activities were all less than 15% that of rats and undetectable in hagfish. CPT/FAO ratios were greater in herring (8.1) and hagfish (greater than 30) than adult rats (3.1), suggesting that peroxisomal beta-oxidation is relatively less important in these species despite a natural diet containing high levels of long chain polyunsaturated fatty acids. These data are discussed in relation to current ideas about the role of peroxisomes in beta-oxidation of fatty acids.     

Metabolic aspects of peroxisomal beta-oxidation

In the course of the last decade peroxisomal beta-oxidation has emerged as a metabolic process indispensable to normal physiology. Peroxisomes beta-oxidize fatty acids, dicarboxylic acids, prostaglandins and various fatty acid analogues. Other compounds possessing an alkyl-group of six to eight carbon atoms (many substituted fatty acids) are initially omega-oxidized in endoplasmic reticulum. The resulting carboxyalkyl-groups are subsequently chain-shortened by beta-oxidation in peroxisomes. Peroxisomal beta-oxidation is therefore, in contrast to mitochondrial beta-oxidation, characterized by a very broad substrate-specificity. Acyl-CoA oxidases initiate the cycle of beta-oxidation of acyl-CoA esters. The next steps involve the bi(tri)functional enzyme, which possesses active sites for enoyl-CoA hydratase-, beta-hydroxyacyl-CoA dehydrogenase- and for delta 2, delta 5 enoyl-CoA isomerase activity. The beta-oxidation sequence is completed by a beta-ketoacyl-CoA thiolase. The peroxisomes also contain a 2,4-dienoyl-CoA reductase, which is required for beta-oxidation of unsaturated fatty acids. The peroxisomal beta-hydroxyacyl-CoA epimerase activity is due to the combined action of two enoyl-CoA hydratases. (For a recent review of the enzymology of beta-oxidation enzymes see Ref. 225.) The broad specificity of peroxisomal beta-oxidation is in part due to the presence of at least two acyl-CoA oxidases, one of which, the trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidase, is responsible for the initial dehydrogenation of the omega-oxidized cholesterol side-chain, initially hydroxylated in mitochondria. Shortening of this side-chain results in formation of bile acids and of propionyl-CoA. In relation to its mitochondrial counterpart, peroxisomal beta-oxidation in rat liver is characterized by a high extent of induction following exposure of rats to a variety of amphipathic compounds possessing a carboxylic-, or sulphonic acid group. In rats some high fat diets cause induction of peroxisomal fatty acid beta-oxidation and of trihydroxy-5 beta-cholestanoyl-CoA oxidase. Induction involves increased rates of synthesis of the appropriate mRNA molecules. Increased half-lives of mRNA- and enzyme molecules may also be involved. Recent findings of the involvement of a member of the steroid hormone receptor superfamily during induction, suggest that induction of peroxisomal beta-oxidation represents another regulatory phenomenon controlled by nuclear receptor proteins. This will likely be an area of intense future research. Chain-shortening of fatty acids, rather than their complete beta-oxidation, is the prominent feature of peroxisomal beta-oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective inhibition of hepatic peroxisomal fatty acid beta-oxidation by enoximone.

Although beta-oxidation of fatty acids occurs in both peroxisomes and mitochondria, beta-oxidizing enzymes in these organelles have distinct differences in their specifity and sensitivity to inhibitors. In this study, the effects of the phosphodiesterase inhibitor enoximone on hepatic peroxisomal and mitochondrial beta-oxidation were investigated. In liver homogenates from control rats, cyanide-insensitive peroxisomal beta-oxidation of palmitoyl-CoA was inhibited progressively by increasing concentrations of enoximone. Similar results were obtained in liver homogenates from rats pretreated with the known peroxisomal proliferator diethylhexylphthalate. In contrast, mitochondrial beta-oxidation of palmitoyl-CoA was not inhibited by enoximone. These data show that enoximone selectively inhibits basal as well as induced peroxisomal, but not mitochondrial, beta-oxidation of the CoA thioester of long-chain fatty acids. The availability of specific inhibitors of peroxisomal beta-oxidation should prove useful in elucidating regulatory mechanisms operative in this pathway in normal as well as in proliferated peroxisomes.     

Biochemical characterization of two functional human liver acyl-CoA oxidase isoforms 1a and 1b encoded by a single gene

Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.     

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ?foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ?scdA?echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ?scdA?echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.     

Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans

In the Ascomycete fungus Aspergillus nidulans, the ratio of conidia (asexual spores) to ascospores (sexual spores) is affected by linoleic acid moieties including endogenous sporogenic factors called psi factors. Deletion of odeA (Delta odeA), encoding a Delta-12 desaturase that converts oleic acid to linoleic acid, resulted in a strain depleted of polyunsaturated fatty acids (18:2 and 18:3) but increased in oleic acid (18:1) and total percent fatty acid content. Linoleic acid-derived psi factors were absent in this strain but oleic acid-derived psi factors were increased relative to wild type. The Delta odeA strain was reduced in conidial production and mycelial growth; these effects were most noticeable when cultures were grown at 26 degrees C in the dark. Under these environmental conditions, the Delta odeA strain was delayed in ascospore production but produced more ascospores than wild type over time. This suggests a role for oleic acid-derived psi factors in affecting the asexual to sexual spore ratio in A. nidulans. Fatty acid composition and spore development were also affected by veA, a gene previously shown to control light driven conidial and ascospore development. Taken together our results indicate an interaction between veA and odeA alleles for fatty acid metabolism and spore development in A. nidulans.