Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate.

摘要: The enzyme catalysing the first step in anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses reaction phenylacetate+CoA+ATP → phenylacetyl-CoA+AMP+PPi and requires Mg2+. Phenylacetate-CoA ligase (AMP forming) found cells grown anaerobically with nitrate. Maximal specific activity 0.048 μmol min-1 x mg-1 protein mid-exponential growth phase. After 640-fold purification 18% yield, 24.4 achieved. is single polypeptide Mr 52 ±2 kDa. shows high specificity towards aromatic inducer substrate uses ATP preferentially; Mn2+ can substitute for apparent Km values phenylacetate, CoA, are 60, 150, 290 μM, respectively. soluble has an optimum pH 8.5, insensitive to oxygen, but rather labile presence glycerol and/or stabilization. N-terminal amino acid sequence showed no homology other reported CoA-ligases. expression enzye studied by immunodetection. present not mandelate, phenylglyoxylate, benzoate; small amounts were detected aerobically phenylacetate.