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Related Experiment Videos

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization.

David B Hawkes1, Gregory W Adams, Alma L Burlingame

  • 1Department of Chemistry, University of Queensland, Brisbane, Queensland 4067, Australia.

The Journal of Biological Chemistry
|May 23, 2002
PubMed
Summary

We discovered a novel cytochrome P450 (P450(cin)) from Citrobacter braakii that metabolizes cineole. This enzyme may use flavodoxin as its redox partner, a first for P450s, and is key to cineole biodegradation.

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Area of Science:

  • Biochemistry
  • Microbiology
  • Enzymology

Background:

  • Cytochromes P450 are crucial hemoproteins for oxidative metabolism in diverse organisms.
  • Bacterial P450 studies offer insights into eukaryotic P450s, bypassing common challenges.
  • Monoterpene oxidation by bacterial P450s is an area of significant research interest.

Purpose of the Study:

  • To characterize a novel cytochrome P450 (P450(cin), CYP176A1) from Citrobacter braakii.
  • To investigate its role in cineole metabolism and biodegradation.
  • To elucidate its unique structural and functional properties, including its redox partners.

Main Methods:

  • Purification of P450(cin) to homogeneity.
  • Amino acid sequencing of tryptic peptides.

Related Experiment Videos

  • PCR-based cloning and sequencing of the cinA gene.
  • Heterologous expression in Escherichia coli.
  • Gas chromatography-mass spectrometry (GC-MS) for product identification.
  • UV-visible spectroscopy for enzyme characterization.

    • Main Results:

    • Isolation and characterization of P450(cin) from Citrobacter braakii, utilizing cineole as a sole carbon source.
    • Identification of a gene cluster encoding P450(cin), a flavodoxin, and a reductase, suggesting flavodoxin as a novel redox partner.
    • Discovery of an unusual active site substitution (asparagine for threonine).
    • Successful heterologous expression and purification of active P450(cin) in E. coli.
    • In vivo formation of 2-hydroxycineole from cineole, indicating P450(cin)'s role in initial monooxygenation.
    • High affinity for cineole (K(D) = 0.7 µM) and characteristic P450 spectral properties.

    Conclusions:

    • P450(cin) is a novel enzyme involved in the initial oxidative metabolism of cineole.
    • This enzyme represents the first characterized P450 system potentially utilizing flavodoxin as its natural redox partner.
    • The findings provide critical insights into bacterial P450 diversity and the biodegradation pathways of monoterpenes.