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 Km values for 4-hydroxybenzoate, ATP, and coenzyme A are 37 microM, 77 microM, and 125 microM, 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 microM). 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-hydroxybenzoate or anaerobically with benzoate or 4-hydroxyphenylacetate.
Biegert
T
T
Abteilung Angewandte Mikrobiologie, Universität Ulm, Germany.
Altenschmidt
U
U
Eckerskorn
C
C
Fuchs
G
G
D000595:Amino Acid Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D010226:Parabens / Q000378:metabolism; D010636:Phenols / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D010226:Parabens; D010636:Phenols; C058481:4-hydroxybenzoate coenzyme A ligase; D003066:Coenzyme A Ligases; C038193:4-hydroxybenzoic acid; D010100:Oxygen