Purification and Characterization of Acinetobacter Calcoaceticus 4-Hydroxybenzoate 3-Hydroxylase after its Overexpression in Escherichia coli

摘要

The enzyme 4-hydroxybenzoate 3-hydroxylase (EC 1.14.13.2) from Acinetobactercalcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pohA) in Escherichia coli. Overexpression was accomplished by placing the lolA 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 lolA and pobA. The product of the overexpressed pobA, 4-hydroxybenzoate hydroxylase, was purified in high yield in two chromatographic steps. The homogeneous protein was 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.