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

Kung, Johannes W., Löffler, Claudia, Dörner, Katerina, Heintz, Dimitri, Gallien, Sébastien, Van Dorsselaer, Alain, Friedrich, Thorsten, Boll, Matthias
Proceedings of the National Academy of Sciences of the United States of America 2009 v.106 no.42 pp. 17687-17692
Geobacter metallireducens, adenosine triphosphate, anaerobes, aromatic compounds, bacteria, chromatography, electron paramagnetic resonance spectroscopy, enzymes, genes, hydrolysis, in vitro studies, iron, sulfur
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.