Engineering of Cytochrome P 450 3 A 4 for Enhanced Peroxide-Mediated Substrate Oxidation Using Directed Evolution and Site-Directed Mutagenesis

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 >6and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a >4and 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 4to 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 6 -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. Mammalian cytochromes P450 comprise a ubiquitous superfamily of heme-containing enzymes, which perform a variety of oxidative reactions on a wide range of substrates including 90% of drugs and environmental pollutants (Coon, 2005). P450-derived metabolites are often biologically active themselves, and understanding their effects is crucial in evaluating a drug’s efficacy, toxicity, and pharmacokinetics. Such studies, however, can require large quantities of the pure metabolites, which may be difficult to synthesize. An alternate approach is to use human P450s to generate the metabolites of drugs and drug candidates. Limitations include poor activity, stability, and expression of P450s in Escherichia coli. In addition, a troublesome reconstitution of P450s, which involves NADPH-cytochrome P450 reductase (CPR), often cytochrome b5 (b5), and phospholipids concurrent with an expensive cofactor, NADPH, further limits their application in synthesis. Therefore, designing xenobiotic-metabolizing P450s having enhanced activity, stability, and expression, and using an alternate oxygen donor such as hydrogen peroxide (H2O2) or cumene hydroperoxide (CuOOH), is highly desirable (Kumar and Halpert, 2005). CYP3A4 is the most abundant P450 enzyme in human liver and metabolizes a wide variety of drugs ( 50%) and carcinogens (Guengerich, 1999; Nebert and Russell, 2002). Because of its pharmacological and environmental significance, CYP3A4 has been the subject of a large number of structure-function studies involving X-ray crystallography, protein modeling, and site-directed mutagenesis (Domanski and Halpert, 2001; Hutzler and Tracy, 2002; Ekins et al., 2003; Tanaka et al., 2004; Williams et al., 2004; Yano et al., 2004; Park et al., 2005; Scott and Halpert, 2005). However, no attempt has been made to engineer CYP3A4 for enhanced catalytic activity, especially in a peroxide system. More recently, microsomal and E. coli-expressed CYP3A4-CPR with an NADPH generating system has been used to produce drug metabolites in milligram quantities (Vail et al., 2005). Over the past decade, directed evolution has been used successfully to design industrial biocatalysts for enhanced catalytic efficiency, stability, and versatility (Schoemaker et al., 2003; Gillam, 2005). Exciting recent results with the bacterial enzyme P450 BM3 have This work was supported by National Institutes of Health Grants GM54995 and Center Grant ES06676. This work was partially presented at Experimental Biology 2006, April 1–5, 2006, San Francisco, CA, and the 5 SW P450 meeting, May 8–10, 2006, Navasota, TX. Article, publication date, and citation information can be found at http://dmd.aspetjournals. org. doi:10.1124/dmd.106. 012054. ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction; 7-BQ, 7-benzyloxyquinoline; 7-BFC, 7-benzyloxy-4-(trifluoromethyl)coumarin; CPR, NADPH-cytochrome P450 reductase; b5, cytochrome b5; HOOH, hydrogen peroxide; CuOOH, cumene hydroperoxide; OH, hydroxy; UTMB, University of Texas Medical Branch. 0090-9556/06/3412-1958–1965$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 12 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 12054/3155225 DMD 34:1958–1965, 2006 Printed in U.S.A. 1958 at A PE T Jornals on July 9, 2017 dm d.aspurnals. org D ow nladed from