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

Fernandez, Javier ; Dimarco, Anthony A. ; Ornston, L. Nicholas ; Harayama, Shigeaki

In: The Journal of Biochemistry, 1995, vol. 117, no. 6, p. 1261-1266

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    Titel
    • Purification and Characterization of Acinetobacter calcoaceticus 4-Hydroxybenzoate 3-Hydroxylase after Its Overexpression in Escherichia coli
    Autor
    • Fernandez, Javier. Department of Medical Biochemistry, University Medical Center 1 rue Michel Servet, 1211 Geneva, Switzerland
    • Dimarco, Anthony A.. Department of Biology, Yale University New Haven, CT 06511, USA
    • Ornston, L. Nicholas. Department of Biology, Yale University New Haven, CT 06511, USA
    • Harayama, Shigeaki. Department of Medical Biochemistry, University Medical Center 1 rue Michel Servet, 1211 Geneva, Switzerland
    Dokumententyp
    • Postprint
    OAI-PMH-ID
    • oai:doc.rero.ch:296520
    Summary
    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