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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.)

(Received July 17, 2006; accepted September 13, 2006)

	Abstract

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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.

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 illustrated the potential of directed evolution for engineering P450s that use artificial oxygen donors (Glieder et al., 2002; Meinhold et al., 2005). Recently, P450 BM3 has been engineered by directed evolution to produce the authentic human metabolites of propranolol (Otey et al., 2006). Directed evolution of several mammalian P450s for enhanced activity has been established (Kim and Guengerich, 2004) and their potential applications have been reviewed recently (Kumar and Halpert, 2005). In addition, we have developed a directed evolution approach to engineer P450 2B1 for enhanced H₂O₂-supported 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (Kumar et al., 2005a). In the present study, we have engineered CYP3A4 using the combination of directed evolution and site-directed mutagenesis for enhanced utilization of peroxide. The most efficient enzyme, F228I/T309A, shows a >11-fold higher k_cat/K_{m, CuOOH} than CYP3A4 for 7-benzyloxyquinoline (7-BQ) debenzylation. In addition, an engineered enzyme, T433S, can metabolize testosterone more efficiently than CYP3A4 and has the potential to produce milligram quantities of 6ß-OH testosterone.

View larger version (44K): [in this window] [in a new window]   FIG. 1. Primers for the construction of CYP3A4 random and site-directed mutants. The codons that were changed to make the desired mutation are shown in bold. 1 These mutants were created by random mutagenesis using error-prone PCR of the cDNA of CYP3A4, as described under Materials and Methods.

	Materials and Methods

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Random Mutagenesis by Error-Prone PCR and Construction of Mutant Libraries. Random mutagenesis was essentially performed using errorprone PCR of the cDNA as described previously for P450 2B1 (Kumar et al., 2005a). Initially, error-prone PCR was standardized to ensure a mutation rate of 1 to 2 base pairs per P450 by using 25 mM MgCl₂, 10 mM dCTP, 10 mM dTTP, and 2 mM each dATP and dGTP (Cirino et al., 2003). Subsequently, ligation and transformation were standardized to obtain >1000 clones per PCR by selecting suitable forward and reverse primers (Fig. 1), restriction sites (NcoI/KpnI), ligation conditions (overnight ligation kit), and transformation procedures (commercial XL-Blue E. coli).

Site-Directed Mutagenesis. T309A, T309I, and T309F were created as described previously (Domanski et al., 2001). Whereas T309V was created using CYP3A4 as a template, other multiple mutants were created using a single/double mutant as a template and appropriate forward and reverse primers as presented in Fig. 1. To confirm the desired mutation and verify the absence of unintended mutations, all constructs were sequenced at the University of Texas Medical Branch Protein Chemistry Laboratory (Galveston, TX).

Screening and Selection. Screening and selection of random mutants were essentially done as described previously for P450 2B1 (Kumar et al., 2005a). Liquid handling was performed using a multichannel robot Biomek 2000 (Beckman Coulter, Fullerton, CA). In brief, transformed E. coli was grown in a 300-µl, V-shaped 96-well microplate containing 150 µl of Luria broth media for 18 to 20 h at 30°C on a microplate shaker. Next, 20 µl of the Luria broth-grown cells were inoculated into a 300-µl, V-shaped 96-well microplate containing 180 µl of Terrific broth medium and grown for 3 h until the cell density reached O.D.₆₀₀ = 1 to 1.5. -Aminolevulinic acid and isopropyl ß-D-thiogalactoside were then added to the cells to final concentrations of 80 and 240 µg/ml, respectively. The cells were then grown for 48 h at 30°C on a microplate shaker. Cells were then harvested by centrifugation at 5000g for 10 min using an Allegra 25R microplate centrifuge (Beckman Coulter). The supernatant was removed by decanting, and the pellet was dried. The cells were resuspended in 150 µl of 0.1 M Hepes buffer, pH 7.4, which is termed whole cells.

Next, 50 µl of the substrate mixture (400 µM 7-BQ containing 4% methanol and 5 U/well polymyxin B sulfate, respectively) was incubated with 40 µl of whole cells for 5 min at room temperature. The background intensity was recorded at _ex = 405 nm and _em = 510 nm using a fluorescence microplate reader (Fluoroskan Ascent; Thermo Electron Corporation, Waltham, MA). The reaction was initiated by the addition of 10 µl of H₂O₂ (10 mM final concentrations), and the formation of product was recorded at 2.5 min. Mutants with 2-fold higher activity than the average template activity were sequenced at the UTMB Protein Chemistry Laboratory and were further characterized as described below.

Expression and Purification of Wild-Type and Engineered Enzymes. CYP3A4 and mutants were expressed as His-tagged proteins in E. coli TOPP3 and purified using a nickel affinity column as described previously (Domanski et al., 2001). Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA). The specific contents of wild-type and most random mutants were between 10 and 15 nmol of P450 per mg of protein. Upon purification, the yield, purity, and stability of the random mutant proteins were either similar to or higher than those of the wild-type enzyme. T309V, T309A/L216W, and F228I/F241V showed low P450 expression (<40 nM), high P420 (>75%), and low specific content (<4.0 nmol of P450 per mg of protein). L216W/T309V and F228I/T309V showed very low P450 expression (>10 nM) and were not purified for further study.

Enzyme Assays. H₂O₂- and CuOOH-supported enzyme activities were assayed as described previously with slight modifications (Kumar et al., 2005a,b). Substrate mixtures were prepared in 100 mM Hepes buffer, pH 7.4, with 2% as the final concentration of methanol (100 µl/well of a 96-well microplate). The substrate mixture was preincubated with 25 or 50 pmol of purified P450 enzyme at room temperature for 5 min. Reactions were initiated by the addition of H₂O₂ (10 mM) or CuOOH (2.5 mM). For steady-state kinetics with the peroxide, different concentrations of H₂O₂ (1–25 mM) or CuOOH (0.05–2.5 mM) were used at 200 µM 7-BQ. After 3 min of incubation, the reactions were stopped by adding 340 units (50 µl) of catalase. Subsequently, 50 µl of 100 mM Hepes buffer, pH 7.4, was added before recording the fluorescence intensity at _ex = 405 nm and _em = 510 nm using an Ascent fluorescence plate reader (Thermo Electron). For 7-BFC O-deethylation and testosterone 6ß-hydroxylation, 75 µM and 200 µM concentrations of the substrates, respectively, were incubated with the enzyme systems before initiating the reactions using appropriate concentrations of CuOOH as described above. Testosterone hydroxylation was assayed by thin-layer chromatography using radiolabeled substrate (Kumar et al., 2005a), whereas 7-BFC oxidation was assayed using a fluorescence method (Domanski et al., 2001). To determine the effect of b₅, a 1:1 molar ratio of P450 and b₅ at 0.25 µM P450 was incubated for 10 min before incubating with substrates.

The standard NADPH-dependent assays with 7-BQ, 7-BFC, and testosterone were essentially carried out as described previously (Domanski et al., 2001; Kumar et al., 2003). The reconstitution system contained CYP3A4, CPR, and b₅ at a ratio of 1:4:2 and included 10 µg of dioleoyl phosphatidylcholine/100-µl reaction volume.

Steady-state kinetic parameters were determined by regression analysis using SigmaPlot (SPSS, Inc., Chicago, IL). The K_m value was determined using the Michaelis-Menten equation, whereas the S₅₀ and n values were determined using the Hill equation. Each kinetic experiment included CYP3A4 and mutants (presented in Tables 1, 2, 3, 4, 5, 6) simultaneously, using a high-throughput microplate assay for more accurate comparison of the data. The standard errors for fit to Michaelis-Menten or Hill equations were large when the K_{m, HOOH} or S_{50, 7-BQ} was higher than the maximum concentration of the reactants used.

P450 Heme-Depletion Assay. Determination of the kinetics of CYP3A4 heme depletion in the presence of CuOOH was done as described previously for H₂O₂ (Kumar et al., 2005b). In brief, the reaction was carried out at 25°C in 100 mM Hepes buffer, pH 7.4, in a 1-ml semi-microspectrophotometric cell with constant stirring. The reaction mixture contained 1 µM P450 and different concentrations of CuOOH (0.01–15 mM). Bleaching of the hemoprotein was followed by measuring a series of absorbance spectra using a Shimadzu-2600 spectrophotometer in the 340- to 700-nm range (Shimadzu, Kyoto, Japan). Fitting of the titration curves was performed by regression analysis using SigmaPlot (SPSS, Inc.). A simple pseudo-first order equation was used to determine the k_inact values at different CuOOH concentrations, and the Michaelis-Menten equation was used to determine the K_{I, CuOOH} value.

	Results

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Directed Evolution of CYP3A4. The most critical step in directed evolution is to find a simple, economical, and high-throughput activity screen. Recently, we have developed a facile assay method to circumvent steps involving CPR and b₅ by using the alternate oxygen donor H₂O₂ for CYP3A4 (Kumar et al., 2005b). Because H₂O₂-supported activity with 7-BQ is very low in the wild type, several existing CYP3A4 mutants were examined. Only L211F/D214E and L373F showed 1.5-fold higher activity than CYP3A4. However, L211F/D214E showed abolished enzyme cooperativity and L373F showed low expression and stability (data not shown). Therefore, CYP3A4 was used as the template for directed evolution. The activity screen was optimized using whole cell suspensions as described earlier for P450 2B1 (Kumar et al., 2005a). A concentration of 10 mM H₂O₂ was used for screening mutants using whole cell suspensions because, above this concentration, the reaction is not linear up to 2.5 min (data not shown). A representative assay of multiple colonies of CYP3A4 indicated 70% colony-to-colony variation in activity (Fig. 2, open triangles) compared with the average CYP3A4 colony. The control showed very low relative intensity (Fig. 2, open diamonds). These data suggested that random clones having 2-fold higher activity than CYP3A4 would correspond to mutants with enhanced catalytic activity.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Development of screening and selection. A, diamonds and triangles represent the activities with 7-BQ determined for individual clones from negative control (plasmid only) and CYP3A4 wild-type, respectively, and are plotted in random distribution of the activity to demonstrate the range across the 96-well microplate. B, the activities of individual clones of randomly mutagenized CYP3A4 are plotted in decreasing order of activity. The x-axis represents individual colonies, and the y-axis represents relative fluorescence intensity for the product, 7-hydroxyquinoline, at ex = 405 nm and em = 510 nm.

Steady-State Kinetics at Varying 7-BQ Concentrations. To test whether the selected purified mutants also showed enhanced k_cat, steady-state kinetic analysis of 7-BQ oxidation at 10 mM H₂O₂ and at varying 7-BQ concentrations was carried out. Above 10 mM, H₂O₂ accelerates heme depletion, and the reaction is not linear up to 5 min (Kumar et al., 2005b). L216W, F228I, and T433S showed 60 to 80% higher k_cat values than did CYP3A4 (Table 1). However, F241V showed only a 20% higher k_cat than did CYP3A4. In addition, F228I showed a significant increase in the S_{50, 7-BQ} and decrease in the n value. Subsequently, to test whether the increased k_cat in the H₂O₂-supported assay is also associated with an increased k_cat in the NADPH-supported reaction, the mutants were assayed in the standard reconstituted system using NADPH. All the mutants showed 10 to 40% lower k_cat than did CYP3A4 with a significant change in the S₅₀ and n values (Table 1). In this assay, the accuracy of the S₅₀ is limited by the highest 7-BQ concentration used. Overall, the results suggested that our screening method is limited to identification of CYP3A4 mutants with enhanced activity in the H₂O₂-supported system.

Steady-State Kinetics at Varying H₂O₂ Concentrations. Subsequently, we sought to determine whether the screening method is targeted to increased k_cat and/or decreased K_{m, HOOH}. Therefore, steady-state kinetic analysis at 200 µM 7-BQ and at varying H₂O₂ concentrations was carried out. At 20 mM H₂O₂, the rate of 7-BQ debenzylation is linear for up to 3 min (data not shown). CYP3A4 showed a very high K_{m, HOOH} (61 mM), which is much higher than the H₂O₂ concentration used for the activity screening (10 mM) (Table 2). None of the mutants showed improvements in k_cat, whereas L216W and F228I showed an 3-fold decrease in the K_{m, HOOH} values, leading to >2.5-fold enhancement in k_cat/K_{m, HOOH} compared with CYP3A4. Compared with CYP3A4, F241V and T433S showed a modest increase in the k_cat/K_{m, HOOH} values as the result of decreased K_{m, HOOH} (Table 2). The results suggested that the screening method is selective for mutants with decreased K_{m, HOOH}. Furthermore, to test the hypothesis that the combination of single mutants would show a further enhancement in k_cat/K_{m, HOOH}, several multiple mutants were constructed. Unexpectedly, these multiple mutants did not show further improvement (Table 2). F228I/T433S and F241V/F228I showed similar k_cat/K_{m, HOOH} values to F228I, whereas most multiple mutants showed decreased k_cat/K_{m, HOOH}, mainly as the result of decreased k_cat.

Steady-State Kinetics at Varying CuOOH Concentrations. Recently, T309V has been shown to display higher activity than CYP2D6 wild-type in a CuOOH-supported reaction (Keizers et al., 2005). Therefore, the existing CYP3A4 mutants T309A, T309I, and T309F were assayed for CuOOH-supported 7-BQ debenzylation. At 200 µM 7-BQ and 2.5 mM CuOOH, T309A showed a >3-fold higher activity, whereas T309I and T309F demonstrated lower activity than CYP3A4. Subsequently, T309V was constructed by site-directed mutagenesis and showed a >5-fold higher activity than CYP3A4. Therefore, steady-state kinetic analysis at 200 µM 7-BQ concentration and at varying CuOOH concentrations was carried out with T309A and T309V. At 2.5 mM CuOOH, the rate of 7-BQ debenzylation is linear for up to 5 min (data not shown). T309A and T309V exhibited a 4- and 6-fold higher k_cat/K_{m, CuOOH}, respectively, than CYP3A4 in the CuOOH-supported reaction, mainly as the result of increased k_cat values (Table 3). When steady-state kinetic analysis was performed in the H₂O₂-supported reaction, T309V exhibited a 2-fold higher k_cat/K_{m, HOOH} than did CYP3A4, whereas T309A showed lower k_cat/K_{m, HOOH} than did CYP3A4 (data not shown). In addition, T309A and T309V showed lower k_cat values in H₂O₂-supported than CuOOH-supported reactions. Furthermore, to test whether T309A and T309V in CYP3A4 follow a trend similar to T309V in CYP2D6, the standard NADPH-supported oxidation of 7-BQ was performed. T309A and T309V showed lower NADPH-supported activity than CYP3A4, as was found in the case of CYP2D6 (data not shown).

Subsequently, we tested individual single random mutants and multiple mutants for CuOOH-supported activity (Table 3). L216W and F228I respectively exhibited >3- and >4.5-fold higher k_cat/ K_{m, CuOOH} than did CYP3A4, whereas F241V and T433S exhibited 2-fold higher k_cat/K_{m, CuOOH} than did CYP3A4. The results indicated that the random mutants that were isolated for decreased K_{m, HOOH} also showed enhanced utilization of CuOOH (k_cat/K_{m, CuOOH}), mainly as the result of increased k_cat values compared with CYP3A4. As seen earlier with the H₂O₂-supported reaction, combination of these single random mutants did not improve the k_cat/K_m for CuOOH further. Therefore, T309A was added to the individual single random mutants to create several multiple mutants, and their activity was assayed in the CuOOH-supported reaction. Among the mutants with reasonable expression (see Materials and Methods), F228I/T309A showed the highest k_cat/K_m for CuOOH, which was 2-fold higher than that of T309A and 11-fold higher than that of CYP3A4 (Table 3). Interestingly, the K_m for CuOOH was lower by more than an order of magnitude than the K_m for H₂O₂ (1.0 mM versus 20–60 mM).

Effect of Cytochrome b₅ on CuOOH-Supported Oxidation of 7-BQ. To investigate whether b₅ also stimulates CuOOH-supported 7-BQ oxidation by CYP3A4, as shown earlier for H₂O₂ (Kumar et al., 2005b), steady-state kinetic analysis was performed at varying CuOOH concentrations and at 200 µM 7-BQ. Cytochrome b₅ stimulated k_cat/K_{m, CuOOH} by 7-fold in CYP3A4, and by >5-fold in random mutants (Table 3 versus Table 4). However, the site-directed mutants T309A and T309V showed relatively less stimulation by b₅. In F228I/T309A, b₅ stimulated k_cat/K_{m, CuOOH} by 3-fold. Interestingly, b₅ stimulated CuOOH-supported k_cat/K_{m, CuOOH} by both increasing the k_cat and decreasing the K_{m, CuOOH} values (Table 3 versus Table 4).

T309A and T309V Exhibit Enhanced P450 3A4 Activity with 7-BFC. The CYP3A4 random mutants were tested with 7-BFC to investigate whether the enhanced CuOOH-dependent activity with 7-BQ also was retained with a structurally distinct substrate. Whereas T309A and T309V showed significantly increased activity with 7-BFC (at 75 µM) by 1.5- and 3.5-fold, respectively, the random mutants showed lower activity than did CYP3A4 (data not shown). Therefore, steady-state kinetic analysis of 7-BFC O-deethylation with CYP3A4, T309A, and T309V was performed. The results are presented in Table 5. It is interesting that the K_{m, CuOOH} for 7-BFC O-deethylation was 5-fold lower than that for 7-BQ debenzylation. T309V showed 2.5-fold higher k_cat/K_{m, CuOOH} for 7-BFC oxidation than did CYP3A4. Furthermore, b₅ stimulated k_cat/K_{m, CuOOH} for the oxidation of 7-BFC by >7-fold for CYP3A4, whereas it stimulated k_cat/K_{m, CuOOH} by 5- and 3-fold for T309A and T309V, respectively (Table 5). The b₅-mediated enhancement of k_cat/K_{m, CuOOH} with 7-BFC was accomplished as the result of increased k_cat and decreased K_{m, CuOOH}. As seen earlier with 7-BQ, T309A and T309V also showed decreased k_cat in the NADPH-supported reaction with 7-BFC compared with CYP3A4 (data not shown).

T433S Exhibits Enhanced Activity with Testosterone. The CYP3A4 random mutants were also assayed with another structurally distinct substrate, testosterone. CYP3A4 yielded 6ß-hydroxytestosterone as the major (92%) and 2ß-hydroxytestosterone as a minor metabolite. The metabolite profile was unchanged in random mutants and T309A, whereas T309V yielded 30% 2ß-hydroxytestosterone. Although most of the mutants showed decreased testosterone 6ß-hydroxylation, T433S showed a >1.5-fold higher activity at 200 µM substrate than did CYP3A4. Therefore, steady-state kinetic analysis of testosterone 6ß-hydroxylation by CYP3A4 and T433S was performed, and the results are presented in Table 6. It is interesting to note that the K_{m, CuOOH} for testosterone hydroxylation was >3-fold lower than that for 7-BQ debenzylation. T433S showed a >3-fold higher k_cat/K_{m, CuOOH} than did CYP3A4 for testosterone 6ß-hydroxylation as the result of increased k_cat and decreased K_{m, CuOOH}. Cytochrome b₅ further stimulated k_cat/K_{m, CuOOH} by >5-fold by increasing the k_cat and decreasing the K_{m, CuOOH} values. In the NADPH system T433S showed a >2-fold higher k_cat than did CYP3A4 with unaltered S_{50, 7-BQ} and n values (Table 6). Other mutants, however, showed either similar or lower k_cat than did CYP3A4 (data not shown). The results suggested that Thr⁴³³ Ser substitution is unique in that it enhances CuOOH- and NADPH-supported reactions with testosterone, but not with 7-BQ or 7-BFC.

Synthesis of 6ß-OH Testosterone by T433S in the Presence of Cytochrome b₅. The decrease in the K_{m, CuOOH} with testosterone T433S and upon b₅ addition is very important, because CuOOH also depletes the heme in a time- and concentration-dependent manner. Therefore, it was critical to find the optimal CuOOH concentrations at which T433S could convert testosterone into 6ß-OH testosterone. CuOOH concentration-dependent heme depletion was examined at different time intervals (1–60 min), and the rate constants for P450 inactivation were determined (Fig. 3). Subsequently, a re-plot of k_inact and CuOOH concentrations using the Michaelis-Menten equation was carried out, which gave a K_{I, CuOOH} of 6.5 mM (Fig. 3). The K_{I, CuOOH} for heme depletion is >10-fold higher than the K_{m, CuOOH} for testosterone hydroxylation. Therefore, lower concentrations (0.1–0.25 mM) of CuOOH should be suitable for longer incubations to achieve nearly complete product formation.

View larger version (26K): [in this window] [in a new window]   FIG. 3. P450 3A4-heme depletion assays at different concentrations of CuOOH and 0.25 µM P450 as described under Materials and Methods. The plot represents the percentage of P450 (y-axis) as a function of incubation time (x-axis) at increasing concentrations of CuOOH. The plots of the fit to a first-order equation yielded kinetic parameters as follows: 0.00 mM (kinact = 0.003 min–1), 0.05 mM (kinact = 0.004 min–1), 0.10 mM (kinact = 0.016 min–1), 0.25 mM (kinact = 0.020 min–1), 0.50 mM (kinact = 0.046 min–1), 2.0 mM (kinact = 0.070 min–1), 5.0 mM (kinact = 0.110 min–1), 10 mM (kinact = 0.180 min–1), and 15 mM (kinact = 0. 203 min–1). The plot is representative of two independent experiments.

A time course experiment with CYP3A4 and T433S in the absence and presence of b₅ containing 50 µM testosterone, 0.25 µM CYP3A4 and b₅, and 0.2 mM CuOOH was carried out (Fig. 4). The results showed that T433S in the presence of b₅ converts 75% of testosterone to 6ß-OH testosterone in 2 h. Conversely, CYP3A4 in the absence of b₅ converts only 5% to 6ß-OH testosterone in 2 h. Furthermore, to test whether the percentage formation of 6ß-OH testosterone was reduced when the reaction volume was increased, 0.1-, 1.0-, and 10-ml reactions were carried out in a container with a large surface area, and with occasional mixing. The results showed that there was only a modest decrease in the percentage conversion when the reaction volume was increased by 100-fold (data not shown).

View larger version (19K): [in this window] [in a new window]   FIG. 4. CuOOH-dependent formation of 6ß-OH testosterone by CYP3A4 and T433S in the absence and presence of cytochrome b5. Reactions were carried out at different time intervals, and product formation was measured as described under Materials and Methods. Error bars represent the mean ± S.D (n = 3).

	Discussion

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View larger version (59K): [in this window] [in a new window]   FIG. 5. Ribbon schematic representation of a three-dimensional structure of CYP3A4. The heme porphyrin (red sticks), heme iron (red sphere), and mutant residues (orange spheres) are shown. The structure was generated using the PDB ID 1TQN (Yano et al., 2004) and PyMol graphics (DeLano, 2004).

The general mechanism by which peroxide supports P450-mediated substrate oxidation remains unclear, because significant activity requires an appropriate cytochrome P450, substrate, and peroxide. An X-ray crystal structure of CYP3A4 suggests that Arg-212 may provide general base catalysis of peroxide cleavage (Yano et al., 2004). Therefore, replacement by a nonbasic residue may decrease or even eliminate such activity. To explore this hypothesis we probed H₂O₂-supported oxidation of 7-BQ by R212A. However, there was no significant effect of this replacement on k_cat (data not shown). Based on the crystal structure of P450eryF T252A, the conserved threonine is thought to be involved in proton delivery to the oxygen species (Raag et al., 1991). It has been hypothesized that P450 can deploy three different oxygenating species (peroxo, hydroperoxo, and oxenoid-iron), and they prefer certain types of reactions (Keizers et al., 2005). Furthermore, based on enhanced CuOOH-supported and decreased NADPH-supported activity by T309A in CYP2D6, it was suggested that the mutant predominantly forms the peroxo and/or hydroperoxo complexes as opposed to oxenoid-iron complex, which is mainly preferred by the NADPH-dependent reaction in the wild type. Similarly, the finding that T309V and T309A in CYP3A4 exhibited increased CuOOH- and decreased NADPH-supported activity compared with CYP3A4 is consistent with the hypothesis that the mutants predominantly form peroxo and/or hydroperoxo complexes. In contrast, the corresponding T302A in CYP2B4 and T303A in CYP2E1 showed decreased peroxide-dependent activity with one substrate but increased activity with another substrate (Vaz et al., 1996, 1998), which is consistent with our observations that CYP3A4 T309V showed decreased CuOOH-supported activity with testosterone, but not with 7-BQ or 7-BFC.

Another important observation in this study suggests that the K_{m, CuOOH} is dependent on the substrate used. In the absence of b₅, the K_{m, CuOOH} is 1.2 mM, 0.22 mM, and 0.38 mM for the oxidation of 7-BQ, 7-BFC, and testosterone, respectively. The K_{m, CuOOH} for P450 is usually associated with the accessibility of CuOOH to the active site heme pocket (Zhang and Pernecky, 1999). Removal of the NH₂-terminal region in P450 2B4 caused a 5-fold decrease in K_{m, CuOOH} for N-methylaniline demethylation, suggesting that the N-terminal region might mask the accessibility of CuOOH in the active site (Zhang and Pernecky, 1999). However, the mechanism by which substrates alter CuOOH accessibility is rather unclear. More recently, our laboratory has proposed that binding of a second molecule of substrate that shows homotropic cooperativity, such as 1-pyrenebutanol, induces a conformational transition in CYP3A4 that appears to close the heme pocket (Davydov et al., 2005; D. Davydov, unpublished observations). This phenomenon does not appear to occur when only one molecule of bromocriptine binds to CYP3A4. Therefore, we speculate that 7-BQ, which shows a higher extent of cooperativity than does 7-BFC or testosterone (Harlow and Halpert, 1998; Domanski et al., 2001) (Table 1), masks the active site for the access of CuOOH leading to an increased K_{m, CuOOH}.

Our results from the peroxide-supported reaction suggest that L216W and F228I play a major role in enhancing catalytic efficiency of 7-BQ debenzylation. X-ray crystal structures (Fig. 5) and extensive site-directed mutagenesis do not predict a role of these nonactive site residues in substrate binding and/or metabolism (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, recent molecular dynamics simulations with the newly developed force field parameters for the heme-thiolate group and its dioxygen adduct show differences in structural and dynamic properties between CYP3A4 in the resting form and its complexes with the substrate progesterone and the inhibitor metyrapone (Park et al., 2005). The results indicated that the broad substrate specificity of CYP3A4 stems from the malleability of a loop (residues 211–218) that resides in the vicinity of the channel connecting the active site and bulk solvent. Interestingly, L216W (F-helix) and F228I (F-G loop) mutations are located in or close to this region, which may cause a conformational transition upon 7-BQ binding leading to enhanced peroxide-supported reactions. Phe-241 is located in the G-helix, and its role in substrate binding or metabolism is not clear. Thr-433 is located near the most conserved region, Cys-442, which ligates the heme iron as the fifth ligand.

Although the role of b₅ as a source of electrons for P450 is well known (Schenkman and Jansson, 2003), increasing evidence points to a modulatory effect of b₅ mediated by a conformational transition in P450 (Reed and Hollenberg, 2003; Yamaori et al., 2003; Yamaguchi et al., 2004). Recently, we have shown that b₅ stimulates H₂O₂-supported 7-BQ debenzylation by CYP3A4 (Kumar et al., 2005b). In addition, an independent finding has shown that b₅ directly induces positive cooperativity and enhances catalytic turnover of CYP3A4 with multiple substrates (Jushchyshyn et al., 2005). In the present study, an 3-fold increase in the k_cat and 2-fold decrease in the K_{m, CuOOH} for the oxidation of 7-BQ, 7-BFC, and testosterone further suggests that b₅ plays a major role in modulating CYP3A4 activity. Earlier, it was proposed that b₅ induces some reorganization in the heme moiety and substrate binding pocket of P450 2B4 that favors the binding of peroxide and benzphetamine (Aplentalina and Davydov, 1997). Therefore, we suggest that b₅ reorganizes the CYP3A4 active site heme pocket, which leads to a decreased K_{m, CuOOH} by favoring the binding of CuOOH. Because the random mutants reside outside of the active and b₅ binding sites (Bridges et al., 1998; Schenkman and Jansson, 2003; Williams et al., 2004; Yano et al., 2004), these mutations do not primarily alter the b₅-P450 interaction. In contrast, a reduced b₅-stimulated P450 activity in T309V is similar to earlier observations that I301F and A305F, which are closest to the heme (Williams et al., 2004; Yano et al., 2004), show impaired b₅-P450 interaction (Kumar et al., 2005b). To further test the hypothesis that the b₅-P450 interaction is impaired in T309V, but not in random mutants, the K_d values for b₅ were determined in CYP3A4, T309V, and F228I at 0.25 µM protein concentration as described previously (Kumar et al., 2005b). The K_d for b₅ in T309V (0.30 µM) was increased by 6-fold compared with CYP3A4 (0.05 µM), whereas the K_d for b₅ in F228I (0.04 µM) was similar to that of CYP3A4 (data not shown).

An interest in producing large quantities of P450-mediated drug metabolites prompted us to find a simple and cost-effective way to synthesize such compounds. In a recent study, microsomal CYP3A4 from human liver or recombinant sources and a NADPH regenerating system have been used to produce 50 mg of 6ß-OH testosterone with 70% conversion to product (Vail et al., 2005). Consistent with the above studies, our data predict that in a 1-liter reaction with CuOOH, T433S in the presence of b₅ can produce 10 mg of 6ß-OH testosterone with 60% conversion to product. Although the yield using CuOOH is lower than the standard method, it is more cost-effective. More recently, CYP2D6 and CYP3A4 have been identified and further optimized to use a selected peroxide donor such as CuOOH with improved activity compared with the standard NADPH reconstitution system (Chefson et al., 2006).

In summary, the directed evolution approach created CYP3A4 mutants with enhanced utilization of peroxide, whereas site-directed mutants T309A and T309V specifically showed enhanced CuOOH-supported activity with 7-BQ and 7-BFC. Combination of the site-directed and random mutants created the most efficient enzyme, F228I/T309A, for peroxide-mediated oxidation of several substrates. Addition of b₅ further stimulated the k_cat/K_m for CuOOH-supported CYP3A4 activity, and the CYP3A4 random mutant T433S showed enhanced CuOOH- and NADPH-supported activity with testosterone. The mutations found in this study may be useful for re-engineering other related mammalian P450s for enhanced utilization of peroxide. In addition, this approach may be used to engineer xenobiotic-metabolizing P450 enzymes for facile synthesis of milligram quantities of specific drug metabolites, agrochemicals, and food ingredients, and for bioremediation.

	Acknowledgments

 
We thank Drs. Frances H. Arnold and Edgardo Farinas from California Institute of Technology (Pasadena, CA) for providing technical details of directed evolution and sharing their unpublished materials. We thank Dr. Dmitri Davydov (UTMB) forexpert suggestions, Dr. B. K. Muralidhara (UTMB) for generating Fig. 5, and You-Qun He (UTMB) for technical support in optimizing error-prone PCR. We also thank Dr. Richard Hodge, Synthetic Organic Chemistry Core Laboratory, UTMB, for synthesizing 7-BQ, and Dr. Kenneth Johnson, Pharmacology and Toxicology, UTMB, for allowing us to use the fluorescence plate reader (Fluoroskan Ascent).

	Footnotes

 
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^th 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.

Address correspondence to: Santosh Kumar, Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031. E-mail: sakumar{at}utmb.edu

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