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

作     者：Sun, Ling Chen, Chong S. Waxman, David J. Liu, Hong Halpert, James R. Kumar, Santosh 

作者机构：Univ Texas Med Branch Dept Pharmacol & Toxicol Galveston TX 77555 USA Boston Univ Dept Biol Boston MA 02215 USA Chinese Acad Sci Shanghai Inst Mat Med Drug Discovery & Design Ctr Shanghai 201203 Peoples R China 

出 版 物：《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 (生物化学与生物物理学集刊)

年 卷 期：2007年第458卷第2期

页      面：167-174页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：NCI NIH HHS [CA49248, R01 CA049248] Funding Source: Medline NIEHS NIH HHS [ES03619, P30 ES006676, R01 ES003619, P42 ES007381, 5 P42 ES07381, ES06676] Funding Source: Medline 

主　　题：cytochrome p450 P4502B11 P450 engineering structure-function relationships site-directed mutagenesis enzyme catalysis 

摘      要：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 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 mu M vs. 18 mu M). 1209A also demonstrated enhanced selectivity for testosterone 16 beta-hydroxylation over 16 alpha-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 16a-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 similar to 4-fold decrease in Km. Introduction of the V183L mutation into full-length P450 21311 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. (c) 2007 Elsevier Inc. All rights reserved.