Analysis of two benzo[a]pyrene-resistant mutants of the mouse hepatoma Hepa-1 P(1)450 gene via cDNA expression in yeast.
Two benzo[a]pyrene-resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa-1 wild-type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full-length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn-414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu-118----Arg-118 and Arg-245----Pro-245, but otherwise to encode the normal (524-residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu-118----Arg-118 mutation alone was found to have negligible effect on AHH activity, while the Arg-245----Pro-245 mutation alone leads to a 2- to 3-fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg-245, and the complementary function of Leu-118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur.
Kimura
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Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892.
Smith
H H
HH
Hankinson
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Nebert
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DW
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004351:Drug Resistance; D005796:Genes / Q000187:drug effects; D008114:Liver Neoplasms, Experimental / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic
D001564:Benzo(a)pyrene; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases