title: Rational Re-Engineering of the O-Dealkylation of 7-Alkoxycoumarin Derivatives by Cytochromes P450 2B from the Desert Woodrat Neotoma lepida.
abstract: On the basis of recent functional and structural characterization of cytochromes P450 2B from the desert woodrat (Neotoma lepida), the 7-alkoxycoumarin and 7-alkoxy-4-(trifluoromethyl)coumarin O-dealkylation profiles of CYP2B35 and CYP2B37 were re-engineered. Point mutants interchanging residues at seven positions in the enzyme active sites were created and purified from an Escherichia coli expression system. In screens for O-dealkylation activity, wild-type CYP2B35 metabolized long-chain 7-alkoxycoumarins but not 7-alkoxy-4-(trifluoromethyl)coumarins or short-chain 7-alkoxycoumarins. Wild-type CYP2B37 metabolized short-chain substrates from both series of compounds. CYP2B35 A367V showed maximal activity with 7-butoxycoumarin as opposed to 7-heptoxycoumarin in the parental enzyme, and CYP2B35 A363I/A367V produced an activity profile like that generated by CYP2B37. CYP2B35 A363I/A367V/I477F showed 7-ethoxycoumarin and 7-ethoxy-4-(trifluoromethyl)coumarin O-dealkylation rates similar to those of CYP2B37 and higher than those of the double mutant. A CYP2B35 septuple mutant retained a CYP2B37-like activity profile. In contrast, the CYP2B37 septuple mutant produced very low rates of O-dealkylation of all substrates. As mutating residue 108 in either enzyme was detrimental, this change was removed from both septuple mutants. Remarkably, the CYP2B35 sextuple mutant produced an activity profile that was a hybrid of that of CYP2B35 and CYP2B37, whereas the CYP2B37 sextuple mutant had almost no O-dealkylation activity. Docking of 7-substituted coumarin derivatives into a model of the CYP2B35 sextuple mutant based on a previous crystal structure of the 4-(4-chlorophenyl)imidazole wild-type complex revealed how the mutant can exhibit activities of both CYP2B35 and CYP2B37.
authors.affiliation: Department of Pharmaceutical Science, University of Connecticut School of Pharmacy , Storrs, Connecticut 06269-3092, United States.
authors.forename: Lu
authors.initials: L
authors.lastname: Huo
authors.affiliation: Department of Pharmaceutical Science, University of Connecticut School of Pharmacy , Storrs, Connecticut 06269-3092, United States.
authors.forename: Jingbao
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Biology, University of Utah , Salt Lake City, Utah 84112, United States.
authors.forename: M Denise
authors.initials: MD
authors.lastname: Dearing
authors.affiliation: Department of Pharmaceutical Sciences, West Virginia University School of Pharmacy , Morgantown, West Virginia 26506, United States.
authors.forename: Grazyna D
authors.initials: GD
authors.lastname: Szklarz
authors.affiliation: Department of Pharmaceutical Science, University of Connecticut School of Pharmacy , Storrs, Connecticut 06269-3092, United States.
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.affiliation: Department of Pharmaceutical Science, University of Connecticut School of Pharmacy , Storrs, Connecticut 06269-3092, United States.
authors.forename: P Ross
authors.identifier: 0000-0002-0264-1704
authors.initials: PR
authors.lastname: Wilderman
mesh_terms: D000478:Alkylation; D000818:Animals; D003374:Coumarins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016205:Sigmodontinae
chemical_list: D003374:Coumarins; D003577:Cytochrome P-450 Enzyme System