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

Author:
Kung, Johannes W., Löffler, Claudia, Dörner, Katerina, Heintz, Dimitri, Gallien, Sébastien, Van Dorsselaer, Alain, Friedrich, Thorsten, Boll, Matthias
Source:
Proceedings of the National Academy of Sciences of the United States of America 2009 v.106 no.42 pp. 17687-17692
ISSN:
0027-8424
Subject:
Geobacter metallireducens, adenosine triphosphate, anaerobes, aromatic compounds, bacteria, chromatography, electron paramagnetic resonance spectroscopy, enzymes, genes, hydrolysis, in vitro studies, iron, sulfur
Abstract:
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.
Agid:
2366390