ENZYMES OF ANAEROBIC METABOLISM OF PHENOLIC-COMPOUNDS - 4-HYDROXYBENZOATE-COA LIGASE FROM A DENITRIFYING PSEUDOMONAS SPECIES

The initial step of anaerobic 4-hydroxybenzoate and 3-hydroxybenzoate degradation was studied in a denitrifying Pseudomonas sp. 4-Hydroxybenzoate and 3-hydroxybenzoate are converted into their coenzyme A (CoA) thioesters by two different specific coenzyme A ligases. 4-Hydroxybenzoate-CoA ligase (AMP-forming) was purified 350-fold. The ligase is active as a monomer of molecular mass 48 kDa, as determined by gel filtration and SDS/PAGE. At a pH optimum of 8.5, the apparent K(m) values for 4-hydroxybenzoate, ATP, and coenzyme A are 37 muM, 77 muM, and 125 muM, respectively. The enzyme reacts specifically with 4-hydroxybenzoate (100%) and 4-aminobenzoate (30%). Other analogues of benzoate, notably 3- or 2-hydroxybenzoate, are inactive, and 2,4-dihydroxybenzoate and 2-hydroxy-4-methylbenzoate act as competitive inhibitors (K(i) = 1 muM). Polyclonal antibodies were raised and used in immunoblot assays to study the regulation of the expression of 4-hydroxybenzoate-CoA ligase. The ligase is synthesized when cells are grown anaerobically with 4-hydroxybenzoate, phenol, or p-cresol; phenol and p-cresol are degraded via 4-hydroxybenzoate. The enzyme is not present in cells grown aerobically with 4-hydroyxbenzoate or anaerobically with benzoate or 4-hydroxyphenylacetate.