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
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|