Functional characterization of two polymorphic variants of human cytochrome P450 1A1 using recombinant protein expression

Abstract

The cytochromes P450 (CYP) constitute a superfamily of heme-thiolate enzymes, which catalyze various phase I metabolism reactions, including hydroxylation of saturated carbon-hydrogen bonds, epoxidation of double bonds, oxidation of heteroatoms, dealkylation reactions, and oxidations of aromatics drugs. CYP1A1 is a member of the CYP1 family, which is expressed at high level in extrahepatic tissues and plays an important role in the metabolism of environmental chemicals and hormones. Genetic polymorphism in the coding sequence of CYP gene influences the metabolism of substrates, bioavailability and the development of toxicity, including adverse drug effects and even cancer susceptibility. The genetic polymorphisms of CYP1A1 containing a G to C transition at nucleotide 184 (results in Ala62Pro substitution) and a G to A transition at nucleotide 134 (results in Gly45Asp substitution), which have been has been found limitedly in East Asian population. Previous case-control study suggested that the genetic polymorphism of Ala62Pro and Gly45Asp loci in CYP1A1 might be related to a higher incidence of lung cancer and uterine leiomyoma development, respectively. However, further studies are required to evaluate the effects of these polymorphisms on the metabolism of xenobiotics and their roles in carcinogenesis. In the present study, the CYP1A1 variant harboring Ala62Pro and Gly45Asp were characterized by using heterologous expression in Escherichia coli (E. coli ) and mammalian cells. E. coli and its membrane fraction expressing the variants (Ala62Pro and Gly45Asp), as well as the purified variant protein, had lower CYP (i.e., holoenzyme) contents than their wild-type (WT) equivalents. However, based on the immunoblot analysis, the total level of CYP1A1 apoprotein in E. coli expressing the variants were similar to that in cells expressing the WT. The membrane expressing the variants and the purified variant proteins had reduced heme contents compared with their WT equivalents. In addition, enhanced supplementation of a heme precursor, δ-aminolevulinic acid, during culture did not increase CYP content in E. coli expressing the variant, but did for the WT. E. coli membrane and mammalian microsomes expressing the variants, as well as purified variant proteins, had reduced ethoxyresorufin O-dealkylation activities benzo[a]pyrene hydroxylation activities, compared with the WT equivalents. These findings led us to hypothesize that the Ala62Pro and Gly45Asp substitution alters the ordered framework of the protein structure and causes a loss of heme incorporation. The elucidation of the protein structure responsible for CYP specificity is of great importance in understanding enzyme functions and mechanisms. Molecular modeling based on three-dimensional structure of CYP1A1(PDB code: 4I8V) suggested that, Ala62 and Gly45 residue are localized in an α-helix A and a proline-rich region at N-terminus of CYP1A1, respectively, and considered one of key residue composing the interaction networks responsible for heme binding. In support, the single amino acid substitution of those two and surrounding residues (interacting residues) dramatically decreased CYP content in E. coli expressing the variants. Taken together, our findings suggest the Ala62Pro and Gly45Asp substitution distorts the structural integrity of CYP1A1, and results in the formation of protein that has a decreased heme-binding affinity and holoenzyme with altered enzymatic characteristics. Thus, the G184C and G134A nucleotide polymorphism responsible for this amino acid variation may have a critical impact on affected individuals phenotype in metabolism.