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Research ArticleArticle

Characterization of Pulmonary CYP4B2, Specific Catalyst of Methyl Oxidation of 3-Methylindole

Brian A. Carr, Swayampakula Ramakanth, Ghazi A. Dannan and Garold S. Yost
Molecular Pharmacology May 2003, 63 (5) 1137-1147; DOI: https://doi.org/10.1124/mol.63.5.1137
Brian A. Carr
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Swayampakula Ramakanth
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Ghazi A. Dannan
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Garold S. Yost
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Abstract

The selective toxicity of chemicals to lung tissues is predominately mediated by the selective expression of certain pulmonary cytochrome P450 enzymes. This report describes the purification, cloning, and characterization of a unique enzyme, CYP4B2, from goat lung. The purified P450 enzyme was isolated by multistep ion exchange chromatography to electrophoretic homogeneity with an apparent molecular mass of 55,000 Da. Western blotting studies demonstrated that CYP4B enzymes were selectively expressed in lung tissues of rabbits, rats, and mice. Two cDNAs, CYP4B2 and CYP4B2v, were cloned from goat lung tissue. CYP4B2 was predicted to be 511 amino acids and approximately 82% similar to the four known CYP4B1 proteins. Concurrently, a variant of the known human CYP4B1 cDNA, that contained a S207 insertion, was cloned from human lung tissue. The modified recombinant goat CYP4B2 was expressed in Escherichia coli and the enzyme catalyzed theN-hydroxylation of the prototypical substrate 2AF. CYP4B2 preferentially dehydrogenated, rather than hydroxylated, the pneumotoxicant 3-methylindole (3MI) (Vmax = 4.61 versus 0.83 nmol/nmol of P450/min, respectively). To investigate the relevance of covalent heme binding of CYP4 enzymes in CYP4B2-mediated metabolism of 3MI, a site-directed mutant (CYP4B2/A315E) was evaluated. The mutation had little effect on the Vmax of either dehydrogenation or hydroxylation but increased theKm, which decreased the catalytic efficiency (V/K) for 3MI. The A315E mutation shifted the absorbance maximum of the enzyme from 448 to 451 nm, suggesting that the electron density of the heme was altered. These results demonstrate that CYP4B2 is highly specific for methyl group oxidation of 3MI, without formation of ring-oxidized metabolites, and seems to be predominately responsible for the highly organ-specific toxicity of 3MI in goats.

Footnotes

  • ↵1 Present address: Regeneron Pharmaceuticals, 777 Old Sawmill River Road, Tarrytown, NY 10591.

  • ↵2 Present address: USEPA, Office of Pesticides Programs, 7509C, 401 M Street, S.W., Washington, DC 20460.

  • ↵3 Accession numbers (GenBank) for the CYP4B enzymes in this article are: goat CYP4B2, AY151046; goat CY4B2 variant,AY151047; human CYP4B1, AY151048; human CYP4B1 variant, AY151049.

  • This work was supported by United States Public Health Service grants HL13645 and HL60143 from the National Heart, Lung, and Blood Institute.

  • Abbreviations:
    P450
    cytochrome P450
    3MI
    3-methylindole
    3MEI
    3-methyleneindolenine
    I3C
    indole-3-carbinol
    3MOI
    3-methyloxindole
    PAGE
    polyacrylamide gel electrophoresis
    2AF
    2-aminofluorene
    RT
    reverse transcription
    PCR
    polymerase chain reaction
    RACE
    rapid amplification of cDNA ends
    NAC
    N-acetylcysteine
    bp
    base pair(s)
    EST
    expressed sequence tag
    • Received September 17, 2002.
    • Accepted January 28, 2003.
  • U.S. Government
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Molecular Pharmacology: 63 (5)
Molecular Pharmacology
Vol. 63, Issue 5
1 May 2003
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Research ArticleArticle

Characterization of Pulmonary CYP4B2, Specific Catalyst of Methyl Oxidation of 3-Methylindole

Brian A. Carr, Swayampakula Ramakanth, Ghazi A. Dannan and Garold S. Yost
Molecular Pharmacology May 1, 2003, 63 (5) 1137-1147; DOI: https://doi.org/10.1124/mol.63.5.1137

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Research ArticleArticle

Characterization of Pulmonary CYP4B2, Specific Catalyst of Methyl Oxidation of 3-Methylindole

Brian A. Carr, Swayampakula Ramakanth, Ghazi A. Dannan and Garold S. Yost
Molecular Pharmacology May 1, 2003, 63 (5) 1137-1147; DOI: https://doi.org/10.1124/mol.63.5.1137
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