Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli

J Biochem. 1995 Jun;117(6):1261-6. doi: 10.1093/oxfordjournals.jbchem.a124853.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 4-Hydroxybenzoate-3-Monooxygenase / chemistry
  • 4-Hydroxybenzoate-3-Monooxygenase / genetics
  • 4-Hydroxybenzoate-3-Monooxygenase / isolation & purification*
  • 4-Hydroxybenzoate-3-Monooxygenase / metabolism*
  • Acinetobacter calcoaceticus / enzymology*
  • Amino Acid Sequence
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Folic Acid Antagonists / pharmacology
  • Gene Expression Regulation, Bacterial
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • NADP / antagonists & inhibitors
  • NADP / metabolism
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Trimethoprim / pharmacology

Substances

  • Enzyme Inhibitors
  • Folic Acid Antagonists
  • Recombinant Proteins
  • NADP
  • Trimethoprim
  • 4-Hydroxybenzoate-3-Monooxygenase
  • Tetrahydrofolate Dehydrogenase