Functional characterization of four allelic variants of human cytochrome P450 1A2

https://doi.org/10.1016/j.abb.2003.11.019Get rights and content

Abstract

Human cytochrome P450 1A2 catalyzes important reactions in xenobiotic metabolism, including the N-hydroxylation of carcinogenic aromatic amines. In 2001, Chevalier et al. [Hum. Mutat. 17 (2001) 355] reported four new P450 1A2 sequence variants in the human population. We have now expressed these variants in Escherichia coli and measured protein expression (optical spectroscopy of holoenzyme and immunoblotting) and bioactivation of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQ (2-amino-2,4-dimethylimidazo[4,5-f]quinoline) in the lacZ reversion mutagenicity test. Enzyme kinetic analyses were performed for N-hydroxylation of five heterocyclic amine substrates and for O-deethylation of phenacetin. The most drastic effect was that of the R431W substitution: no holoenzyme was detectable. This residue is located in the “meander” peptide region and earlier site-directed mutagenesis studies demonstrated that it is critical for maintenance of protein tertiary structure. The other three variants had subtly different catalytic activities compared to the wild-type enzyme.

Section snippets

Chemicals

Glu-P-1, IQ, MeIQ, N-hydroxy-MeIQ, nitro-IQ, and PhIP were purchased from Toronto Research Chemicals (Toronto, ON). [Ring -3H]-Phenacetin was a gift of Dr. F.F. Kadlubar (National Center for Toxicological Research, Jefferson, AR). IPTG, δ-ALA, and ampicillin were obtained from Sigma Chemical (St. Louis, MO). Other chemicals were of the highest grades commercially available.

Bacterial culture medium and molecular biology reagents

Bacto agar, bacto yeast extract, and peptone were purchased from Difco (Detroit, MI). Luria–Bertani medium (capsule

Site-directed mutagenesis

All four P450 1A2 variants were successfully constructed by PCR site-directed mutagenesis of the bicistronic plasmid expression vector. In each case, the complete P450 1A2 open reading frame and the tac-tac promoter regions were sequenced, to ensure that no additional mutations had been introduced.

Recombinant protein expression

Immunoblot analysis (Fig. 1) revealed that the levels of expression of P450 1A2 variants C406Y, D348N, and I386F were lower than that of the wild-type enzyme. Variant R431W, however, was undetectable,

Acknowledgements

This study was supported by NIH Grants R01 CA90426 and P30 ES00267 and by the Natural Sciences and Engineering Research Council of Canada.

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