Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G
Abteilung Angewandte Mikrobiologie, Universität Ulm, Germany.
Eur J Biochem. 1993 Apr 1;213(1):555-61. doi: 10.1111/j.1432-1033.1993.tb17794.x.
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.
在反硝化假单胞菌中研究了厌氧条件下4-羟基苯甲酸酯和3-羟基苯甲酸酯降解的初始步骤。4'-羟基苯甲酸酯和3-羟基苯甲酸酯通过两种不同的特异性辅酶A连接酶转化为它们的辅酶A（CoA）硫酯。4-羟基苯甲酸-CoA连接酶（生成AMP）被纯化了350倍。通过凝胶过滤和SDS/PAGE测定，该连接酶以分子量为48 kDa的单体形式具有活性。在最适pH 8.5时，4-羟基苯甲酸、ATP和辅酶A的表观Km值分别为37 μM、77 μM和125 μM。该酶与4-羟基苯甲酸（100%）和4-氨基苯甲酸（30%）特异性反应。苯甲酸的其他类似物，特别是3-或2-羟基苯甲酸酯无活性，2,4-二羟基苯甲酸酯和2-羟基-4-甲基苯甲酸酯作为竞争性抑制剂（Ki = 1 μM）。制备了多克隆抗体并用于免疫印迹分析，以研究4-羟基苯甲酸-CoA连接酶表达的调控。当细胞在厌氧条件下以4-羟基苯甲酸、苯酚或对甲酚生长时，会合成该连接酶；苯酚和对甲酚通过4-羟基苯甲酸降解。在以4-羟基苯甲酸进行需氧生长或在厌氧条件下以苯甲酸或4-羟基苯乙酸生长的细胞中不存在该酶。
