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J. Biol. Chem., Vol. 283, Issue 30, 20713-20721, July 25, 2008

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Aromatizing Cyclohexa-1,5-diene-1-carbonyl-Coenzyme A Oxidase

CHARACTERIZATION AND ITS ROLE IN ANAEROBIC AROMATIC METABOLISM^*

Bärbel Thiele, Oliver Rieder, Nico Jehmlich^¶, Martin von Bergen^¶, Michael Müller, and Matthias Boll¹

From the Institute of Biochemistry, University of Leipzig, Leipzig D-04103, Germany, Institute of Pharmaceutical and Medical Chemistry, University of Freiburg, Freiburg D79104, Germany, and the Department of Proteomics, ^¶Helmholtz Centre for Environmental Research-UFZ,Leipzig D-04318, Germany

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive dearomatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M_r 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K_m = 10 µM) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H₂O. DCO was highly specific for 1,5-dienoyl-CoA (K_m = 27 µM). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile dearomatization/rearomatization reactions catalyzed by BCR and DCO.

Received for publication, April 14, 2008 , and in revised form, May 19, 2008.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant BO 1565/5-1/5-2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute of Biochemistry, Brüderstr. 34, D-04103 Leipzig, Germany. Fax: 49-341-9736919; E-mail: boll{at}uni-leipzig.de.

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