Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases.
Proc Natl Acad Sci U S A
; 106(42): 17687-92, 2009 Oct 20.
Article
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| MEDLINE
| ID: mdl-19815533
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 (K(m) = 24 +/- 4 microM). 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 alpha(2)beta(2)-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The alphabeta 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 alphabeta 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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Tungsteno
/
Geobacter
/
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Año:
2009
Tipo del documento:
Article
País de afiliación:
Alemania