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Journal of Bacteriology, October 2003, p. 6119-6129, Vol. 185, No. 20
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.20.6119-6129.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

2-Oxoglutarate:NADP+ Oxidoreductase in Azoarcus evansii: Properties and Function in Electron Transfer Reactions in Aromatic Ring Reduction

Christa Ebenau-Jehle,1 Matthias Boll,1 and Georg Fuchs1*

Mikrobiologie, Institut für Biologie II, Universität Freiburg, Freiburg, Germany1

Received 10 April 2003/ Accepted 31 July 2003

The conversion of [14C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying ß-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min-1 mg-1. 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH (~10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP+, and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 ± 20 kDa and was composed of three subunits of 63 ({gamma}), 62 ({alpha}), and 37 (ß) kDa in a 1:1:1 ratio, suggesting an ({alpha}ß{gamma})2 composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP+ and oxidized viologens as electron acceptors; in contrast NAD+ was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP+, depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The {alpha}-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the ß-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the {gamma}-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP+-specific KGOR. A similar NADP+-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

* Corresponding author. Mailing address: Mikrobiologie, Institut für Biologie II, Universität Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany. Phone: 49 7612032649. Fax: 49 7612032626. E-mail: georg.fuchs{at}biologie.uni-freiburg.de.

Journal of Bacteriology, October 2003, p. 6119-6129, Vol. 185, No. 20
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.20.6119-6129.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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