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Engineering of Cytochrome P450 3A4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas (S.K., J.R.H.); and the Center for Drug Discovery and Design, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (H.L.)

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 K_{m, HOOH} and a 2.5-fold increase in k_cat/K_{m, 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 k_cat/K_{m, CuOOH} than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a >4- and a >3-fold higher k_cat/K_{m, 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 k_cat/K_{m, CuOOH} than CYP3A4. Addition of cytochrome b₅, which is known to stimulate peroxide-supported activity, enhanced the k_cat/K_{m, CuOOH} of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced k_cat/K_{m, 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 6ß-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.

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