Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate |
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Authors: | Ulrich Härtel Elke Eckel Jürgen Koch Georg Fuchs Dietmar Linder Wolfgang Buckel |
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Affiliation: | (1) Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, W-3550 Marburg, Federal Republic of Germany;(2) Angewandte Mikrobiologie, Universität Ulm, W-7900 Ulm, Federal Republic of Germany;(3) Biochemisches Institut, Fachbereich Humanmedizin, Justus-Liebig-Universität, W-6300 Giessen, Federal Republic of Germany |
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Abstract: | Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%). |
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Keywords: | Glutaryl-CoA dehydrogenase Glutaconyl-CoA decarboxylase Pseudomonas sp. Phototrophic proteobacteria Anaerobic degradation of benzoate FAD Ferricenium |
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