Fernandez J, Dimarco A A, Ornston L N, Harayama S
Department of Medical Biochemistry, University Medical Center, Geneva, Switzerland.
J Biochem. 1995 Jun;117(6):1261-6. doi: 10.1093/oxfordjournals.jbchem.a124853.
4-Hydroxybenzoate 3-hydroxylase [EC 1.14.13.2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their response to Cl- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined.
通过在大肠杆菌中使该基因（pobA）40倍过表达，将来自乙酸钙不动杆菌的4-羟基苯甲酸3-羟化酶[EC 1.14.13.2]纯化至同质。过表达是通过将folA基因（编码对甲氧苄啶耐药的二氢叶酸还原酶）直接置于pobA基因的下游，并要求重组体在升高浓度的甲氧苄啶上生长来实现的。据推测，存活的变体经历了基因改变，从而使得folA和pobA都能过表达。4-羟基苯甲酸3-羟化酶通过两步色谱法进行纯化，进行了生化特性鉴定，并将其特性与其来自荧光假单胞菌的同源物的特性进行了比较。这两种酶对Cl-离子抑制的反应不同。推测在假定的NADPH结合位点的单个氨基酸变化可解释这种差异。还研究了底物类似物的抑制和催化特性。
