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 micromol 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 (alphabeta)(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 ( approximately 10 microM) 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.
journal_name
J Bacterioljournal_title
Journal of bacteriologyauthors
Dörner E,Boll Mdoi
10.1128/jb.184.14.3975-3983.2002keywords:
subject
Has Abstractpub_date
2002-07-01 00:00:00pages
3975-83issue
14eissn
0021-9193issn
1098-5530journal_volume
184pub_type
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journal_title:Journal of bacteriology
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doi:10.1128/jb.169.3.1315-1320.1987
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pub_type: 杂志文章
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journal_title:Journal of bacteriology
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journal_title:Journal of bacteriology
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pub_type: 杂志文章
doi:10.1128/JB.116.2.915-924.1973
更新日期:1973-11-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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journal_title:Journal of bacteriology
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更新日期:1976-07-01 00:00:00
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journal_title:Journal of bacteriology
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journal_title:Journal of bacteriology
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journal_title:Journal of bacteriology
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更新日期:1999-02-01 00:00:00
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