Polymorphic Rescue of a Human Cytochrome P450 Pseudogene (CYP2B7) Reveals Functional Enzyme Activity in a Subset of Individuals

Cytochrome P450 (CYP) 2B7 is currently classified as a pseudogene due to the presence of a premature stop codon in exon 7 that is predicted to result in a truncated protein. We hypothesized that in some individuals, CYP2B7 may code for a full length protein with enzyme activity similar to CYP2B6, as the result of a DNA sequence polymorphism in the CYP2B7 premature stop codon. Genomic DNA was prepared from our human liver bank (2 Hispanic, 4 African-Americans, and 47 Caucasians) and blood obtained from 20 African-Americans undergoing an unrelated clinical study. PCR was used to amplify and sequence the genomic region surrounding the CYP2B7 premature stop codon. All European-American and Hispanic DNA showed no variation of the stop codon sequence (TGA). In 9 of 24 African Americans, the first nucleotide of the stop codon was heterozygous (T/C), and in one of 24 African-American this nucleotide was homozygous (C/C) resulting in an arginine (codon CGA) instead of the stop codon. We next cloned the CYP2B7-R378 full-length cDNA by reverse transcription PCR from human liver mRNA. However, in addition to the CYP2B7-R378 cDNA, we also cloned a chimeric CYP2B6/CYP2B7-R378, which like CYP2B7-R378 was also predicted to code for a full-length protein. Both CYP2B7-R378 and CYP2B6 amino acid sequences could be aligned to the rabbit CYP2B4 crystal structure indicating that CYP2B7-R378 contained no mutations that would disrupt protein structure. Expression of cDNAs encoding CYP2B7-R378, CYP2B6/CYP2B7-R378, and CYP2B6 in 3T3 fibroblast cells showed immunodetectable CYP2B protein for each, but minimal (CYP2B6) or no bupropion hydroxylation activity. However, by use of the baculovirus-insect cell expression system we successfully showed bupropion hydroxylation activity in cells infected with CYP2B7-R378 and CYP2B6, but not in negative controls (including CYP2B7-X378). No activity was found for chimeric CYP2B6/CYP2B7-R378, although this result needs to be substantiated by further study. Expression of CYP2B7 mRNA was also demonstrated in most human tissues that also contained CYP2B6 mRNA, with particularly high expression in lung, liver and intestine. In conclusion, results indicate that CYP2B7 is expressed as an active cytochrome P450 in some individuals, particularly those of African-American ancestry. The presence of this polymorphic enzyme may explain some important differences in drug response and carcinogen activation related to race and/or ethnicity.