Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modiWed construct
termed P450 2B11dH and characterized for enzyme catalysis using Wve substrates. Mutant I209A demonstrated a 3.2-fold enhanced
kcat/Km for 7-ethoxy-4-triXuoromethylcourmarin O-deethylation, largely due to a dramatic decrease in Km (0.72 M vs. 18 M). I209A
also demonstrated enhanced selectivity for testosterone 16-hydroxylation over 16-hydroxylation. In contrast, V183L showed a 4-fold
increased kcat for 7-benzyloxyresoruWn debenzylation and a 4.7-fold increased kcat/Km for testosterone 16-hydroxylation. V183L also
displayed a 1.7-fold higher kcat/Km than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a
»4-fold decrease in Km. Introduction of the V183L mutation into full-length P450 2B11 did not enhance the kcat/Km. Overall, the
re-engineered P450 2B11dH enzymes exhibited enhanced catalytic eYciency with several substrates including the anti-cancer prodrugs.