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Journal of Bacteriology, July 2002, p. 3975-3983, Vol. 184, No. 14
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.14.3975-3983.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Properties of 2-Oxoglutarate:Ferredoxin Oxidoreductase from Thauera aromatica and Its Role in Enzymatic Reduction of the Aromatic Ring

Edith Dörner and Matthias Boll*

Institut für Biologie II, Mikrobiologie, Universität Freiburg, D-79104 Freiburg, Germany

Received 23 January 2002/ Accepted 26 April 2002

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 µmol min-1 mg-1. The native enzyme had a molecular mass of 200 ± 20 kDa (mean ± standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an ({alpha}ß)2 composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 ± 0.5 mol of Fe, 7.2 ± 0.5 mol of acid-labile sulfur, and 1.6 ± 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low Km for ferredoxin (~10 µM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between 14CO2 and C1 of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S]1+/2+ clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.

* Corresponding author. Mailing address: Institut für Biologie II, Mikrobiologie, Universität Freiburg, Schänzlestr.1, D-79104 Freiburg, Germany. Phone: 49 7612032685. Fax: 49 7612032626. E-mail: boll{at}uni-freiburg.de.

Journal of Bacteriology, July 2002, p. 3975-3983, Vol. 184, No. 14
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.14.3975-3983.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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