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

Abstract

ABSTRACT The conversion of [ 14 C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying β-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 (γ), 62 (α), and 37 (β) kDa in a 1:1:1 ratio, suggesting an (αβγ) 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 α-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the β-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the γ-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 .