Cytochromes P450 2B1 and 2B6: Structure-function analysis using DNA shuffling mutagenesis

The cytochromes P450 (CYP) enzymes are notable for having a wide range of substrates, including endogenous lipids, hormones, environmental chemicals, and pharmaceuticals. Individual CYPs may be highly similar in their DNA sequence and protein structure. However, amino acid sequence differences in the regions that constitute the CYP enzyme active site help define the unique substrate specificity of each individual CYP enzyme. Previous studies have demonstrated that CYP enzyme specificity can be modified by altering these sequence regions. DNA shuffling mutagenesis is a method by which two or more genes having a high sequence identity are randomly cleaved into fragments using DNase I and recombined in vitro using the polymerase chain reaction to produce a library of unique and diverse cDNAs with sequence contributions from more than one of the parental genes. DNA shuffling mutagenesis has been used to create a library of hybrid cytochromes P450 derived from rat CYP2B1 and human CYP2B6. DNA sequence analysis of twenty-three randomly selected cDNA clones from this library revealed 23 novel and unique sequences, with 3.6 +/- 1.4 shuffle events (crossovers) and 10.4 +/- 5.2 point mutations per cDNA, distributed widely through the shuffled cDNAs. Three of the shuffled cDNAs contained point mutations that introduce a premature stop codon, one of which was expressed as a stable truncated peptide. Western blot analysis of the expressed shuffled cDNAs revealed CYP2B immunoreactive proteins for 18 of the 23 cDNAs, although in many cases the CYP2B protein yield was low in isolated microsomes. The high rate of spontaneous mutation may contribute to CYP2B protein instability and/or low recovery in the microsomal fraction. Metabolic studies using cultured cells demonstrated CYP metabolic activity of five of 23 screened shuffled CYP2B enzymes (17%) with the anticancer drugs cyclophosphamide and ifosfamide, and two with the steroid hormone androstenedione. All of the shuffled proteins were apparently less active than the naturally occurring parental CYP2B enzymes.