Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Abstract

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