Re-engineering cytochrome P450 2B11dH 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 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.
Sun
Ling
L
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031, USA.
Chen
Chong S
CS
Waxman
David J
DJ
Liu
Hong
H
Halpert
James R
JR
Kumar
Santosh
S
D019943:Amino Acid Substitution; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D003520:Cyclophosphamide / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006900:Hydroxylation; D007069:Ifosfamide / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000378:metabolism; D051381:Rats; D013739:Testosterone / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D003374:Coumarins; D010078:Oxazines; D011355:Prodrugs; C056033:7-ethoxy-4-trifluoromethylcoumarin; D013739:Testosterone; C057754:benzyloxyresorufin; D003520:Cyclophosphamide; D001189:Aryl Hydrocarbon Hydroxylases; D007069:Ifosfamide