A single acyl-CoA dehydrogenase is required for catabolism of isoleucine, valine and short-chain fatty acids in Aspergillus nidulans

Abstract

An acyl-CoA dehydrogenase has been identified as part of the mitochondrial beta-oxidation pathway in the ascomycete fungus Aspergillus nidulans. Disruption of the scdA gene prevented use of butyric acid (C(4)) and hexanoic acid (C(6)) as carbon sources and reduced cellular butyryl-CoA dehydrogenase activity by 7.5-fold. While the mutant strain exhibited wild-type levels of growth on erucic acid (C(22:1)) and oleic acid (C(18:1)), some reduction in growth was observed with myristic acid (C(14)). The DeltascdA mutation was found to be epistatic to a mutation downstream in the beta-oxidation pathway (disruption of enoyl-CoA hydratase). The DeltascdA mutant was also unable to use isoleucine or valine as a carbon source. Transcription of scdA was observed in the presence of either fatty acids or amino acids. When the mutant was grown in medium containing either isoleucine or valine, organic acid analysis of culture supernatants showed accumulation of 2-oxo acid intermediates of branched chain amino acid catabolism, suggesting feedback inhibition of the upstream branched-chain alpha-keto acid dehydrogenase.

Figure 1

Reactions of fatty acid β-oxidation. Fatty acyl-CoAs are first oxidized to enoyl-CoAs; depending on subcellular localization the electrons are passed either to ubiquinone (via electron transfer flavoproteins in the mitochondria) or to oxygen (producing hydrogen peroxide in peroxisomes). Enoyl-CoAs are hydrated to hydroxyacyl-CoAs, which are in turn oxidized to ketoacyl-CoAs (this time electrons are passed to NAD). The acetyl group is then transferred to a free CoA molecule (release of acetyl-CoA). The remaining acyl-CoA, now shorter by two carbon units, may undergo additional rounds of β-oxidation. Gene products whose functions have been previously described (Maggio-Hall and Keller, 2004) or described in this study are indicated.

Figure 2

Growth of A. nidulans strains on fatty acids as sole carbon source. A. Dry cell weight of mycelium was measured after 72 h incubation in liquid minimal media containing lactose, erucic acid (C_22:1, black bar), oleic acid (C_18:1, grey bar) or hexanoic acid (C₆, white bar). Cultures for each strain were set up in triplicate. Yields for the fatty acids are presented as a percent of the yield for lactose for the indicated strain (WT, A26; ΔscdA, TLMH17). Error bars represent standard error and include error from measurement of the lactose cultures. B. Colony diameter after 96 h incubation on solid minimal medium containing myristic acid, as a percent of the diameter on medium containing lactose (WT, A36; ΔscdA, TLMH17). Spores (10⁵) were inoculated at the center of a standard Petri plate containing 25 ml solid medium. Plates were inoculated in triplicate. Error bars represent standard deviation and include error from measurement of diameters with both lactose and myristic acid. C. Dry cell weight of mycelium was measured after 72 h incubation in minimal medium containing oleic acid. Cultures for each strain were set up in quintuplicate (ΔechA, RLMH41; ΔscdA, TLMH17; ΔechA ΔscdA, RLMH63). Error bars represent standard error.

Figure 3

Growth of wild-type and ΔscdA strains on branched-chain amino acids as sole carbon source. Plates containing 30 ml solid medium were inoculated at the center with 5 x 10⁶ spores and incubated at 37°C for 9 days (Ile and Leu media) or 10 days (Val medium). After two days, plates were sealed with one layer of parafilm to prevent dessication. Shown are: (A) TLMH17 (ΔscdA) and (B) A26 (wild type) inoculated on isoleucine medium, (C) TLMH17 and (D) A26 grown on leucine medium, and (E) TLMH17 and (F) A26 on valine medium. The dark color indicates successful formation of green conidia, the result of asexual development in A. nidulans.

Figure 4

Transcriptional control of scdA by fatty acids and amino acids. Shown is a Northern analysis of scdA transcript levels in the wild-type strain (A26) after 6 h incubation in minimal media containing the following carbon sources: lactose, acetate, hexanoic acid (C6), oleic acid (C18:1), Met, Val, and Ile. Total RNA was isolated from two biological replicates for each carbon source in this analysis. Ethidium bromide-stained rRNA is shown as a control for total RNA loading.

Figure 5

Accumulation of intermediates of branched-chain amino acid catabolism in the ΔscdA mutant, TLMH17, incubated with Val. A. Detection of intermediates of Val, Ile, and Leu catabolism in culture supernatants of wild type and mutant (ΔscdA) strains of A. nidulans: 2-oxoisovaleric acid, 2-oxo-3-methylvaleric acid, 2-oxoisocaproic acid, and 3-hydroxyisobutyric acid. 2-oxocaproic acid was added to culture supernatants as an internal standard prior to sample lyophilization and derivatization. Shown are normalized GC/MS peak area measurements for wild type (A26, dark bars) and ΔscdA mutant (TLMH17, light bars). The 2-oxo acids were normalized using the peak areas of the 2-oxocaproic acid internal standard. The 3-hydroxyisobutyric acid peak areas were normalized using the 4-nitrophenol internal standard. B. Shown is the proposed Val catabolic pathway in A. nidulans, based on studies in other eukaryotes (Bachhawat et al., 1957; Robinson and Coon, 1957) and genetic and genomic data for A. nidulans (Maggio-Hall and Keller, 2004). Valine is converted to 2-oxoisovaleric acid by the action of an unidentified transaminase. 2-Oxoisovaleric acid is activated by the branched-chain α-keto acid dehydrogenase (BCKAD, Pettit et al., 1978) to yield a branched-chain acyl-CoA, isobutyryl-CoA. This is converted to 3-hydroxyisobutyryl-CoA by two enzymes of β-oxidation, acyl-CoA dehydrogenase and enoyl-CoA hydratase. After release of CoA, 3-hydroxyisobutyrate is expected to be converted to methylmalonate semialdehyde and then to propionyl-CoA and CO₂. Propionyl-CoA is metabolized for energy via the methylcitric acid cycle in A. nidulans (Brock et al., 2000).

Figure 6

Accumulation of intermediates of branched-chain amino acid catabolism in the ΔscdA mutant incubated with Ile. A. Detection of intermediates of Val, Ile, and Leu catabolism in culture supernatants of wild type and mutant (ΔscdA) strains of A. nidulans: 2-oxoisovaleric acid, 2-oxo-3-methylvaleric acid, and 2-oxoisocaproic acid. 2-oxocaproic acid was added to culture supernatants as an internal standard prior to sample lyophilization and derivatization. Shown are normalized GC/MS peak area measurements for wild type (A26, dark bars) and ΔscdA mutant (TLMH17, light bars). B. Shown is the proposed Ile catabolic pathway in A. nidulans, based on studies in other eukaryotes (Robinson et al., 1956) and genetic and genomic data for A. nidulans (Maggio-Hall and Keller, 2004). Ile is converted to 2-oxo-3-methylvaleric acid by the action of an unidentified transaminase. Branched-chain α-keto acid dehydrogenase (BCKAD) then generates 2-methylbutyryl-CoA, a substrate for β-oxidation enzymes, including ScdA (as indicated). One complete oxidative cycle yields acetyl-CoA and propionyl-CoA.