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

Abstract

ABSTRACT 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 (αβ) 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 K m for ferredoxin (∼10 μM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between 14 CO 2 and C 1 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.