Re-engineering cytochrome P450 2B 11 dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide
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 2B11 dH and characterized for enzyme catalysis using tive substrates. Mutant I209A demonstrated a 3.2-fold enhanced Kcat/Km for 7-ethoxy-4-tritluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in Km (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 Кcat for 7-bcnzyloxyresorufin debenzylation and a 4.7-fold increased Kcat/Km for testosterone 16α-hydroxylation. V 183L 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 2B11 dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.
Cytochrome P450 P450 2B11 P450 engineering Structure-function relationships Site-directed mutagenesis Enzyme catalysis
Ling Sun Chong S.Chen David J.Waxman Hong Liu James R.Halpert Santosh Kumar
Department of Pharmacology and Toxicology, University of Texas Medical Branch.301 University Blvd., Department of Biologv, Boston University, 5 Cummington Street.Boston.MA 02215, USA Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Drug Discovery and Design Center,
国际会议
广州
英文
22-29
2008-11-01（万方平台首次上网日期，不代表论文的发表时间）