Enzymes of anaerobic metabolism of phenolic compounds

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Abstract
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 μM, 77 μM, and 125 μM, 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 μM). 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.