Abstract
Different anaerobic bacteria can oxidize a variety of aromatic compounds completely to CO2 via one common aromatic intermediate, benzoyl-CoA. It has been postulated that anaerobically the aromatic nucleus of benzoyl-CoA becomes reduced. An oxygen-sensitive enzyme system is described catalyzing the reduction of benzoyl-CoA to trans-2-hydroxycyclohexanecarboxyl-CoA in a denitrifying Pseudomonas species grown anaerobically on benzoate plus nitrate. The assay mixture consists of cell extract, [U-14C]benzoyl-CoA, a [U-14C]benzoyl-CoA-generating system (consisting of [U-14C]benzoate, purified benzoate-CoA ligase, Mg(2+)-ATP, coenzyme A), an ATP-regenerating system (consisting of phosphoenolpyruvate, pyruvate kinase, myokinase), and a low-potential reductant [titanium(III) citrate]. The optimal pH is about 7, the specific activity 10 nmol benzoyl-CoA reduced min-1 x mg-1 protein. The apparent Km for benzoyl-CoA is below 50 microM. Five major products were found. One product is cyclohex-1-enecarboxyl-CoA which must have been formed by a benzoyl-CoA reductase. The other product is probably trans-2-hydroxycyclohexanecarboxyl-CoA rather than the cis-stereoisomer; this product must have been formed by a cyclohex-1-enecarboxyl-CoA hydratase. Two other products are likely to be intermediates of benzoyl-CoA reduction to cyclohex-1-enecarboxyl-CoA, suggesting that the reduction reaction is more complex. An early formed fifth product is more polar than cyclohexanecarboxyl- or cyclohex-1-enecarboxyl-CoA. The enzyme system is under oxygen control since it was not found in cells grown aerobically on benzoate. It is induced by aromatic compounds since its activity is low in cells grown anaerobically on acetate. The actual inducer is probably benzoyl-CoA rather than benzoate. This conclusion is drawn from the fact that the system is also present in cells grown anaerobically on phenol, phenylacetate, 4-hydroxybenzoate, or 2-aminobenzoate; the anaerobic metabolism of these compounds has been shown in this organism to proceed directly via benzoyl-CoA rather than via free benzoate.
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