Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a > 4-fold decrease in differential inhibition by BIF and 4-CPI (IC50,BIF/IC50,4-CPI). F296A, T302A, I363A, V367A, and V477A showed a ⩾2-fold decreased kcat for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered Km. T302A, V367L, and V477A showed > 4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a ⩾150-fold enhanced kcat/Km with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.