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Acetyl-coenzyme A hydrolase, an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase.

ABSTRACT: 1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.

SUBMITTER: Costa ND

PROVIDER: S-EPMC1172457 | biostudies-other | 1975 Nov

REPOSITORIES: biostudies-other

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Project description:Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues, AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus Despite the essentiality of AfERG10B having been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly, AfERG10A localizes to the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus The reduced expression of Aferg10A leads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure of AfERG10A and its complex with CoA are solved, revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline.IMPORTANCE A growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus Current therapeutic options rely on a limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.

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Acetyl-coenzyme A hydrolase, an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase.

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