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

Arch Biochem Biophys. 2007 Feb 15;458(2):167-74. doi: 10.1016/ Epub 2007 Jan 8.


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 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-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 16alpha-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 approximately 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Antineoplastic Agents, Alkylating / metabolism*
  • Aryl Hydrocarbon Hydroxylases / genetics*
  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Coumarins / metabolism
  • Cyclophosphamide / metabolism*
  • Directed Molecular Evolution*
  • Hydroxylation
  • Ifosfamide / metabolism*
  • Liver / enzymology
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Oxazines / metabolism
  • Oxidation-Reduction
  • Prodrugs / metabolism
  • Rats
  • Testosterone / metabolism


  • Antineoplastic Agents, Alkylating
  • Coumarins
  • Oxazines
  • Prodrugs
  • 7-ethoxy-4-trifluoromethylcoumarin
  • Testosterone
  • benzyloxyresorufin
  • Cyclophosphamide
  • Aryl Hydrocarbon Hydroxylases
  • Ifosfamide