Identification, characterization, and site-directed mutagenesis of recombinant pentachlorophenol 4-monooxygenase

Biochim Biophys Acta. 2004 Aug 2;1700(2):151-9. doi: 10.1016/j.bbapap.2004.04.008.


In a previous study, we constructed a three-dimensional (3D) structure of pentachlorophenol 4-monooxygenase (PcpB). In this study, further analyses are performed to examine the important amino acid residues in the catalytic reaction by identification of the proteins with mass spectrometry, circular dichroism (CD) and UV spectrometry, and determination of kinetic parameters. Recombinant histidine-tagged PcpB protein was produced and shown to have a similar activity to the native protein. Mutant proteins of PcpB were then produced (F85A, Y216A, Y216F, R235A, R235E, R235K, Y397A and Y397F) on the basis of the proposed 3D structure. The CD spectra of the proteins showed that there were no major changes in the structures of the mutant proteins, with the exception of R235E. Steady-state kinetics showed a 20-fold reduction in k(cat)/K(m) and a ninefold increase in K(m) for Y216F and a threefold reduction in k(cat)/K(m) and a sixfold increase in K(m) for Y397F compared to the wild type. On the other hand, the value of k(cat)/K(m) of R235K mutant was the same as that of wild type. As a result, it was confirmed that Y216 and Y397 play an important role with respect to the recognition of the substrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Binding Sites / genetics
  • Catalytic Domain / genetics
  • Cloning, Molecular
  • Kinetics
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / metabolism
  • Mutagenesis, Site-Directed*
  • Protein Conformation
  • Recombinant Proteins
  • Spectrum Analysis


  • Bacterial Proteins
  • Recombinant Proteins
  • Mixed Function Oxygenases
  • pentachlorophenol monooxygenase