Details

Autor(en) / Beteiligte

• Kung, Johannes W
• Löffler, Claudia
• Dörner, Katerina
• Heintz, Dimitri
• Gallien, Sébastien
• Van Dorsselaer, Alain
• Friedrich, Thorsten
• Boll, Matthias

Titel

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2009-10, Vol.106 (42), p.17687-17692

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2009

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (Km = 24 ± 4 μM). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73- and 20-kDa subunits, suggesting an α₂β₂-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The αβ unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S]⁰/⁺¹ and three [4Fe-4S]⁺¹/⁺² clusters per αβ unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W- cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.