Structural and functional characterization of "laboratory evolved" cytochrome P450cam mutants showing enhanced naphthalene oxygenation activity

Koji Matsuura, Takehiko Tosha, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Isao Morishima

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H 2O 2, only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron.

Original languageEnglish
Pages (from-to)1209-1215
Number of pages7
JournalBiochemical and biophysical research communications
Volume323
Issue number4
DOIs
Publication statusPublished - 2004 Oct 29
Externally publishedYes

Keywords

  • C-H bond activation
  • Cytochrome P450
  • Dioxygen
  • Electronic absorption
  • Gas chromatographic analysis
  • Hydrogen peroxide
  • Laboratory evolution
  • Naphthalene oxidation
  • P420 form
  • Resonance Raman

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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