Characterization of an acyl-CoA: carboxylate CoA-transferase from Aspergillus nidulans involved in propionyl-CoA detoxification.
Filamentous fungi metabolize toxic propionyl-CoA via the methylcitrate cycle. Disruption of the methylcitrate synthase gene leads to an accumulation of propionyl-CoA and attenuates virulence of Aspergillus fumigatus. However, addition of acetate, but not ethanol, to propionate-containing medium strongly reduces the accumulation of propionyl-CoA and restores growth of the methylcitrate synthase mutant. Therefore, the existence of a CoA-transferase was postulated, which transfers the CoASH moiety from propionyl-CoA to acetate and, thereby, detoxifying the cell. In this study, we purified the responsible protein from Aspergillus nidulans and characterized its biochemical properties. The enzyme used succinyl-, propionyl- and acetyl-CoA as CoASH donors and the corresponding acids as acceptor molecules. Although the protein displayed high sequence similarity to acetyl-CoA hydrolases this activity was hardly detectable. We additionally identified and deleted the coding DNA sequence of the CoA-transferase. The mutant displayed weak phenotypes in the presence of propionate and behaved like the wild type when no propionate was present. However, when a double-deletion mutant defective in both methylcitrate synthase and CoA-transferase was constructed, the resulting strain was unable to grow on media containing acetate and propionate as sole carbon sources, which confirmed the in vivo activity of the CoA-transferase.
Fleck
Christian B
CB
Leibniz Institute for Natural Product Research and Infection Biology e.V., Hans Knoell Institute, Beutenbergstr. 11a, D-07745 Jena, Germany.
Brock
Matthias
M
D000214:Acyl Coenzyme A / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000254:growth & development; D002244:Carbon / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015256:Coenzyme A-Transferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005816:Genetic Complementation Test; D007700:Kinetics; D010641:Phenotype; D017384:Sequence Deletion; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D018076:DNA, Complementary; D005656:Fungal Proteins; C009061:propionyl-coenzyme A; D002244:Carbon; D002950:Citrate (si)-Synthase; D015256:Coenzyme A-Transferases; C092884:methylcitrate synthase