Purification and Characterization of Acinetobacter calcoaceticus 4-Hydroxybenzoate 3-Hydroxylase after Its Overexpression in Escherichia coli

摘要

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 reponse to Cl− ion inhibition. A single ami no 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