Severe Inhibition of Long-Chain Acyl-CoA Enoylhydratase (EC 4.2.1.74) in a Newborn Foal Suffering From Atypical Myopathy

In horses, congenital defects of energy production from long-chain fatty acids have not been described so far. In contrast, inhibition of fatty acid degradation caused by the toxins hypoglycin A and methylenecyclopropylglycine from various maple species are observed frequently. These non-proteinogenic aminoacids are passed on placentally to fetuses or with collostrum or milk to newborn foals. Nevertheless, newborn foals become very rarely symptomatic. Vertical transmission apparently is not sufficient to induce clinical disease without a particular genetic constellation being present. One of these rare cases was investigated here using samples from a mare and her foal. Intoxication by hypoglycin A and methylenecyclopropylglycine is also of interest to human pathology, because these toxins have caused fatal poisonings after consumption of certain fruits many times, especially in children. Maple toxins, their metabolites and some short-chain acyl compounds were quantified by ultrahigh-pressure liquid chromatography/tandem mass spectrometry. An comprehensive spectrum of long-chain acylcarnitines was prepared using electrospray ionization tandem mass spectrometry. Organic acids and acylglycines were determined by gas chromatography mass spectrometry. For evaluation, results of other horses poisoned by maple material as well as unaffected control animals were used. In the serum of the foal, hypoglycin A was detected at a low concentration only. Toxin metabolites reached <3.5% of the mean of a comparison group of horses suffering from atypical myopathy. The spectrum of acylcarnitines indicated enzyme inhibition in short-chain and medium-chain regions typical of acer poisoning, but the measured concentrations did not exceed those previously found in clinically healthy animals after maple consumption. The values were not sufficient to explain the clinical symptoms. In contrast, a remarkably strong enrichment of tetradecenoylcarnitine and hexadecenoylcarnitine was observed. This proves a blockade of the long-chain enoyl-CoA hydratase (EC 4.2.1.74). Vertical transfer of maple toxins to a newborn foal is sufficient for induction of clinical disease only if there is an additional specific reactivity to the active toxins. This was found here in an inhibition of long-chain enoyl-CoA hydratase. Isolated dysfunction of this enzyme has not yet been reported in any species. Further studies are necessary to prove a specific genetic defect.

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic β-oxidation

Abstract2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

Crystal structure of enoyl-CoA hydratase from Thermus thermophilus HB8

Fatty-acid degradation is an oxidative process that involves four enzymatic steps and is referred to as the β-oxidation pathway. During this process, long-chain acyl-CoAs are broken down into acetyl-CoA, which enters the mitochondrial tricarboxylic acid (TCA) cycle, resulting in the production of energy in the form of ATP. Enoyl-CoA hydratase (ECH) catalyzes the second step of the β-oxidation pathway by the syn addition of water to the double bond between C2 and C3 of a 2-trans-enoyl-CoA, resulting in the formation of a 3-hydroxyacyl CoA. Here, the crystal structure of ECH from Thermus thermophilus HB8 (TtECH) is reported at 2.85 Å resolution. TtECH forms a hexamer as a dimer of trimers, and wide clefts are uniquely formed between the two trimers. Although the overall structure of TtECH is similar to that of a hexameric ECH from Rattus norvegicus (RnECH), there is a significant shift in the positions of the helices and loops around the active-site region, which includes the replacement of a longer α3 helix with a shorter α-helix and 310-helix in RnECH. Additionally, one of the catalytic residues of RnECH, Glu144 (numbering based on the RnECH enzyme), is replaced by a glycine in TtECH, while the other catalytic residue Glu164, as well as Ala98 and Gly141 that stabilize the enolate intermediate, is conserved. Their putative ligand-binding sites and active-site residue compositions are dissimilar.

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic β-oxidation

ABSTRACT2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).