Enzymes of anaerobic metabolism of phenolic compounds: 4-hydroxybenzoate-CoA ligase from a denitrifying Pseudomonas species
1993
Biegert, T. | Altenschmidt, U. | Eckerskorn, C. | Fuchs, G.
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 Km values for 4-hydroxybenzoate, ATP, and coenzyme A are 37 micromolar, 77 micromolar, and 125 micromolar, 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 (Ki = 1 micromolar). 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.
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