Background The Cytochromes P450 2B6 (CYP2B6) and 2A6 (CYP2A6) participate in the metabolism of the anti‐HIV drug Efavirenz (EFV). CYP2B6 monohydroxylation of EFV results in the formation of 8‐hydroxyefavirenz (8‐OHEFV) whereas CYP2A6 monohydroxylation of EFV forms 7‐hydroxyefavirenz (7‐OHEFV). To date, little is known about the structural characteristics of these enzymes that lead to differential EFV metabolite formation. In order to better understand such characteristics, we sought to mutate CYP2B6 towards CYP2A6 in hopes of uncovering amino acids critical for the formation of 8‐OHEFV. Specifically, we decided to focus on two phenylalanine residues in the CYP2A6 active site that are valines at the analogous positions in CYP2B6. Methods The valine residues at positions 104 and 477 of CYP2B6 were mutated to phenylalanines using site directed mutagenesis, creating a V104F/V477F double mutant. Wild type (WT) and mutant CYP2B6 expression was accomplished in COS7 cells and, following treatment with EFV, metabolites were extracted and detected using a novel gas chromatography‐mass spectrometry (GC‐MS) method after chemical derivatization using BSTFA + 10% TMCS. Protein expression was assessed via western blotting. Results We observed little to no difference in the protein expression of WT and mutant CYP2B6 proteins. 8‐OHEFV production in the COS7 system was detected for wild type and mutant CYP2B6, but no 7‐OHEFV production was observed. Interestingly, GC‐MS analysis of 8‐OHEFV formation by the V104F/V477F mutant indicates a marked increase in 8‐OHEFV production, suggesting the combination of these mutations might enhance 8‐OHEFV formation by CYP2B6. Future Goals Future experiments will seek to characterize each single mutant and determine kinetic parameters for 8‐OHEFV formation by all three mutant CYP2B6 proteins. Support or Funding Information Funding for this work was generously provided by the Department of Biology and Chemistry at Azusa Pacific University and the Faculty Research Council of Azusa Pacific University
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