title: Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.
abstract: CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031, USA. sakumar@utmb.edu
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D001555:Benzene Derivatives / Q000737:chemistry; D003374:Coumarins / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006861:Hydrogen Peroxide / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011804:Quinolines / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: C419147:7-benzyloxy-4-trifluoromethylcoumarin; D001555:Benzene Derivatives; D003374:Coumarins; D011804:Quinolines; C065956:7-benzyloxyquinoline; D013739:Testosterone; D006861:Hydrogen Peroxide; D051544:Cytochrome P-450 CYP3A; C007164:cumene hydroperoxide