Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

Abstract

A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3–mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.

Keywords

cytochrome P450 monooxygenase BM3 CYP BM3 continuous-flow enzyme activity detection organic solvent tolerability flow-injection analysis

Introduction

Cytochrome P450 monooxygenases (CYPs) are a family of proteins that catalyze a multitude of reactions.¹ These reactions include hydroxylations, epoxidations, dealkylations, reductions, and other oxidative reactions and are performed on substrates that range from alkanes to complex endogenous molecules. CYPs can introduce atomic oxygen into allylic positions, double bonds, and nonactivated C-H bonds.² CYPs are the most important enzymes in human drug metabolism, and it is estimated that approximately 75% of all drugs are metabolized by one or more CYPs.³ During development of novel drugs, investigation of the metabolic fate of drug candidates is crucial because drug metabolites produced by CYPs may have improved pharmacological activities or may be responsible for toxicity or unwanted pharmacological side effects associated with the parent drug. Therefore, systems are needed that enable facile (bio)synthesis of sufficient quantities of drug metabolites for structural elucidation and pharmacological and toxicological evaluation.

One of the most promising CYPs to be used as a biocatalyst for metabolite production is the microbial CYP BM3 (CYP102A1) from Bacillus megaterium. CYP BM3 is the most active CYP identified thus far, and it is catalytically self-sufficient because it is a natural fusion protein containing the heme domain and reductase on a single polypeptide chain.^4,5 Wild-type CYP BM3 is involved in metabolism of long-chain fatty acids and cannot metabolize xenobiotics such as drugs. Rational design and directed evolution approaches have been employed to generate CYP BM3 mutants that can be used for production of human relevant metabolites.^6–8 These mutant enzymes can perform regio- and stereoselective bioconversions very efficiently, especially in buffer, at room temperature and under atmospheric pressure. However, many substrates in industrial processes have low water solubility; therefore, organic solvents are often supplemented to enhance solubility and to increase the enzyme-turnover rate. Addition of organic solvents can lead to destabilization of the enzymes or to a strong reduction of enzymatic activity.^9,10 Therefore, there is a need to improve organic solvent tolerability of CYP BM3 mutants and to understand underlying mechanisms of effects of organic solvents on CYP BM3. In 2004, a study was published describing creation and identification of CYP BM3 mutants with improved organic solvent tolerability.¹¹

This study presents a novel setup to investigate the organic solvent tolerability of CYP BM3 mutants. A recently published online screening method to screen for diversity within CYP BM3 mutant libraries¹² has been adapted to create a platform that allows online screening of CYP BM3 activities at different organic solvent concentrations. Furthermore, the setup was altered in such a way that activity of CYP BM3 mutants against an organic solvent gradient can be investigated. The different setups were used to investigate the organic solvent tolerability of a small library of already existing and novel CYP BM3 mutants. The combination of site-directed mutations T235A, R471A, E494K, and S1024E was selected because this was previously shown to improve organic solvent tolerance of CYP BM3 F87A toward DMSO, acetone, acetonitrile (ACN), ethanol, and tetrahydrofuran.¹¹ These mutations were applied to drug-metabolizing mutants M01, M02, M05, and M11 as templates. We demonstrate here that online screening can be used to study the organic solvent tolerability of CYP BM3 mutants.

Materials and Methods

Chemicals

Benzyloxyresorufin and allyloxyresorufin were synthesized as described previously.^12,13 All other reagents and chemicals were of analytical grade and were purchased from standard commercial suppliers unless stated otherwise.

Enzymes and Plasmids

Bacterial CYP BM3 mutants M01, M02, M05, and M11 in the pET28a+ vector were described previously.¹⁴ The first series of triple mutants (MT11, MT112, MT113, and MT114) each contained the R471A, E494K, and S1024E mutations introduced in M01, M02, M05, and M11, respectively. The second series of quadruple mutants (MT133, MT134, MT135, and MT136) contained an additional T235A mutation introduced in the triple mutants. Supplementary Table S1 tabulates all mutations present in the CYP BM3 mutants compared with wild-type CYP BM3. The mutations were introduced into the corresponding templates in the pBluescript II KS(+) vector using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The forward primers used are described in Supplementary Table S2. Reverse primers were exactly complementary to the forward primers. After mutagenesis, the presence of the desired mutations was confirmed by DNA sequencing (Service XS, Leiden, The Netherlands). The genes were cloned from the pBluescript II KS(+) system, where they reside between the BamHI/EcoRI restriction sites, into the pET28a+ vector as described previously.¹⁵ The resulting His-tagged CYP BM3 mutants MT111, MT112, MT113, MT114, MT133, MT134, MT135, and MT136 were used in this study.

M01, M02, M05, and M11 mutant proteins were expressed as described previously.¹⁶ pET28a+ constructs of all other mutants used were transformed into Escherichia coli BL21 (DE3) cells using standard procedures. For expression, 600 mL Terrific Broth (TB) medium (24 g/L yeast extract, 12 g/L tryptone, and 20 mL/L glycerol) with 30 µg/mL kanamycin was inoculated with 15 mL of an overnight culture. Cells were grown at 175 rpm and 37 °C until OD₆₀₀ reached 0.6. Protein expression was then induced by the addition of 0.6 mM isopropyl-β-d-thiogalactopyrasonide. The temperature was lowered to 20 °C, and 0.5 mM of the heme precursor δ-aminolevulinic acid was added. Expression was allowed to proceed for 18 h. Cells were harvested by centrifugation (4600 × g, 4 °C, 25 min), and the pellet was resuspended in 20 mL KPi-glycerol buffer (100 mM potassium phosphate, pH 7.4, 10% glycerol, 0.5 mM EDTA, and 0.25 mM dithiothreitol). Cells were disrupted using an EmulsiFlex-C3 (Avestin Inc., Ottawa, ON, Canada; 1000 psi, 3 repeats), and the cytosolic fraction was separated from the membrane fraction by ultracentrifugation of the lysate (120.000 × g, 4 ºC, 60 min). CYP concentrations were determined using the carbon monoxide (CO) difference spectrum assay as described by Omura and Sato.¹⁷

Instrumentation

Two Knauer K-500 high-performance liquid chromatography (HPLC) pumps (Berlin, Germany) were used to control 120-mL superloops, made in house, containing enzyme (CYP BM3) and cofactor/substrate (reduced form of nicotinamide-adenine dinucleotide phosphate [NADPH]/alkoxyresorufin), respectively. Two Knauer K-500 HPLC pumps were used for control of the organic solvent concentration in the carrier flow and for delivery of injected samples. CYP BM3 and NADPH/alkoxyresorufin solutions were prepared in potassium phosphate buffer (100 mM; pH 7.4). Both superloops were kept on ice. Flow restrictors were inserted between the pumps and superloops to ensure proper operation of the pumps at low flow rates. Flow restrictors consisted of natural peek tubing and gave back pressures of approximately 50 bar. Pressure limits of the pumps were set 20 bar higher than the working pressure, and VICI Jour backpressure regulators (Schenkon, Switzerland) were inserted after the superloops to prevent damage as a result of possible clogging of the system. For injections of samples, a Gilson 234 autoinjector (Villiers-le-Bel, France) equipped with a Rheodyne (Bensheim, Germany) six-port injection valve (injection loop, 10 μL) was used. To maintain reaction coils at a constant temperature, a Shimadzu CTO-10AC column oven (Duisburg, Germany) was integrated into the system. An Agilent 1100 (Waldbronn, Germany) series fluorescence detector (λ_ex 530 nm; λ_em 580 nm) was used for monitoring fluorescence. An Agilent 1100 series ultraviolet (UV) detector (340 nm) was used for monitoring NADPH consumption. All hardware was integrated into one system by Kiadis B.V. (Groningen, The Netherlands) and was controlled by software developed by Kiadis B.V.

Metabolism of Alkoxyresorufins by CYP BM3 Mutants

Enzyme activities of the CYP BM3 mutants toward benzyloxyresorufin and allyloxyresorufin were measured according to the method of Burke and Mayer¹³ with modifications. These two alkoxyresorufins were selected because they generally showed the highest rate of resorufin production when incubated with CYP BM3 mutants.^12,20 Specific activities were determined by measuring resorufin formation off-line on a Shimadzu RF-1501 spectrofluorometer set at λ_ex 530 nm and λ_em 586 nm. All measurements were performed in duplicate at 24 ºC in a total volume of 1 mL, and 100 mM potassium phosphate buffer (pH 7.4) was used for all experiments. CYP BM3 mutants were introduced in the assay in buffer at a final concentration of 20 nM. Alkoxyresorufin stock solutions were prepared in DMSO and alkoxyresorufins were introduced into the assay for 30 s of preincubation at a final concentration of 10 µM (2% DMSO). Reactions were initiated by addition of 100 µL of 1 mM NADPH, resulting in a final concentration of 100 µM. The increase in resorufin fluorescence was measured over 2 min and the initial linear increases in resorufin fluorescence were used to quantify enzymatic activities.

CYP BM3 Enzyme Activity Screening in Flow-Injection Analysis Mode

A schematic overview of the enzyme activity detection (EAD) system in flow-injection analysis (FIA) mode is depicted in Supplementary Figure S1. The flow from a superloop containing CYP BM3 is continuously mixed with the flow from a superloop containing a mixture of substrate (10 µM benzyloxyresorufin) and cofactor (400 µM NADPH). Samples (10 µL) are introduced by flow injection into a carrier solution at a flow rate of 200 µL/min. Injected compounds and CYP BM3, added to the carrier stream via an inverted Y-type mixing union at a flow rate of 100 µL/min, are allowed to bind in a knitted 0.5-mm i.d. polytetrafluoroethylene (PTFE) reaction coil (50 µL). This mixture is combined with the flow of the benzyloxyresorufin/NADPH solution (100 µL/min). After mixing in a knitted 0.5-mm i.d. PTFE reaction coil (250 µL), benzyloxyresorufin is converted into the highly fluorescent product resorufin. Constant temperature of both reaction coils is maintained at 24 °C.

To test the activity toward the substrate benzyloxyresorufin at fixed percentages of organic solvent, a slightly different EAD setup was used. The superloop containing CYP BM3 was replaced by a loop containing only potassium phosphate buffer (100 mM; pH 7.4). Different CYP BM3 mutants (10 pmol) were introduced by flow injection into a carrier solution consisting of a fixed percentage of organic solvent (the total flow rate was 200 µL/min). Activity of the different CYP BM3 mutants toward benzyloxyresorufin (10 µM) was determined by quantifying the fluorescent signal of the corresponding resorufin product peak formed. NADPH consumption was determined by quantifying the decrease of the UV signal measured at 340 nm.

CYP BM3 Enzyme Activity Screening in Gradient Mode

Activity toward benzyloxyresorufin at varying organic solvent concentrations was investigated by screening benzyloxyresorufin O-dealkylating activity against a gradient of methanol (MeOH). Instead of using a carrier solution with a fixed percentage of MeOH, the two pumps controlling the carrier flow were used to run a gradient of MeOH at a total flow rate of 200 µL/min. The gradient applied was as follows: constant for 5 min at 1% of MeOH, linear increase from 1% to 49% of MeOH, and constant for 5 min at 49% of MeOH. Activity of the different CYP BM3 mutants (50 nM) toward benzyloxyresorufin (10 µM) was determined by monitoring the fluorescent signal of the resorufin product formed. NADPH (100 μM) consumption was monitored by following the decrease of the UV signal measured at 340 nm.

CYP BM3–Mediated Metabolism of the Drug Amitriptyline

Metabolic incubations for the drug amitriptyline were performed in 100 mM potassium phosphate buffer (pH 7.4) with the cytosolic fraction containing 250 nM of the different CYP BM3 mutants at a 100 µM substrate concentration. The final volume of the incubations was 250 µL and the final DMSO concentration varied from 1% to 30%. Reactions were initiated by the addition of 50 µL NADPH, resulting in a final concentrations of 100 μM. The reaction was allowed to proceed for 30 min at 24 °C and was terminated by the addition of 500 µL ice-cold ACN. After centrifugation at 14,000 rpm for 10 min, 5 µL supernatant was analyzed by liquid chromatography (LC)–mass spectrometry (MS). All reactions were performed in duplicate.

Extracts were separated by reversed-phase chromatography using a C18 column (Luna C18[2], 5 μm, 4.6 × 150 mm i.d.; Phenomenex, Amstelveen, The Netherlands) at a flow rate of 0.4 mL/min and a temperature of 25 °C. The gradient was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN). The gradient applied was as follows: constant at 5% B for 1 min, linear increase from 5% B to 40% B in 19 min, linear increase from 40% B to 50% B in 0.1 min, constant for 2.8 min, and linear decrease to 5% B in 0.1 min. The column was allowed to re-equilibrate for 12 min at 5% B. Samples were analyzed on an Agilent 1200 Series Rapid resolution LC device equipped with a Time-of-Flight (TOF) Agilent 6230 mass spectrometer. The mass spectrometer was operated at a capillary voltage of 3500 V with nitrogen as the drying gas (12 L/min) and nebulizer gas (pressure 60 psig). The gas temperature was 350 ºC during operation. The TOF mass spectrometer was used in the positive mode and data were acquired using Mass Hunter workstation software (version B.06.00; Agilent Technologies). Standard curves of the substrates were linear between 1 and 250 µM (data not shown).

Results and Discussion

This study aimed to develop an alternative approach to screen CYP BM3 mutants for organic solvent tolerability. Therefore, a recently developed continuous-flow bioassay setup that has been successfully used to screen CYP BM3 libraries for diversity was adapted.

Expression and Activity of Novel CYP BM3 Mutants

The first step was to create novel CYP BM3 mutants with increased organic solvent tolerability by introducing mutations previously described by Wong et al.¹¹ Although expression yields varied among the different mutants (data not shown), at least 50 nmol of CYP BM3 protein was expressed per 600 mL of TB culture for each mutant. The CO-difference spectra did not show a peak at 420 nm, indicating that the mutations introduced did not affect the stability of the CYP BM3 mutants. Cytosolic fractions were used to prepare 5 μM of CYP BM3 stock solutions in KPi-glycerol buffer for all mutants.

Activities of the four templates and the eight novel mutants toward benzyloxyresorufin and allyloxyresorufin were determined in an off-line fashion to determine which of the two alkoxyresorufins showed the highest O-dealkylation activity. All mutants displayed activity toward the two substrates, but benzyloxyresorufin O-dealkylation (BROD) activity was generally higher than allyloxyresorufin O-dealkylation activity (Suppl. Fig. S2). It was therefore decided to use benzyloxyresorufin as substrate in the FIA mode and gradient studies.

For M11, a BROD activity of 5.83 nmol resorufin/min per nmol CYP was detected, which corresponds very well with the value reported previously by Vottero et al.¹⁸ (5.43 nmol resorufin/min per nmol CYP). For M01, M02, and M11, introduction of the combined R471, E494K, and S1024E mutations resulted in a decrease in BROD activity. For M05, introduction of these mutations resulted in only a slight increase in activity. Introduction of the additional T235A mutation resulted in a significant decrease in activity for MT133 and MT135 compared with their templates MT111 and MT113, respectively. For MT134 and MT136, no significant effect on activity was observed compared with their templates MT112 and MT114, respectively.

CYP BM3 Activity Screening in FIA Mode

It was shown previously that the FIA EAD system provided in Supplementary Figure S1 can be used to screen activity of different CYP BM3 mutants using alkoxyresorufins as fluorogenic substrate.¹² The carrier solution that was used initially consisted of phosphate buffer only. All 12 CYP BM3 mutants (10 pmol) were injected in duplicate and resorufin formation and NADPH consumption were monitored. As shown in Figure 1 , both BROD activity and NADPH consumption could be measured simultaneously. The activity profile generated for the different mutants corresponds very well with the activity profile generated during the off-line BROD activity measurements. Calibration curves were made for resorufin and NADPH (data not shown) to calculate the amounts of resorufin that were formed and the amounts of NADPH that were consumed (see Suppl. Table S3). Overall precision of the screening assay for measuring BROD activity and NADPH consumption was high, as variability of duplicate experiments was less than 4% and 7%, respectively. HPLC analyses of off-line BROD incubations showed no other products besides resorufin (data not shown). Therefore, BROD coupling efficiencies were determined by quantifying resorufin formation and NADPH consumption. It was found that coupling efficiencies were very low, as they ranged between 0.3% and 1.6%. For M11, a coupling efficiency of 0.99% was determined, which agrees with the efficiency of 0.75% reported by Vottero et al.¹⁸ for M11-mediated BROD.

Figure 1.

(A and B) Benzyloxyresorufin O-dealkylation (BROD) activities (A) and BROD-induced NADPH consumption (B) measured in the enzyme activity detection setup. Different BM3 mutants (10 pmol) were introduced by flow injection into a carrier solution consisting of buffer only. The injection order of the mutants (duplicate injections) was as follows: M01, MT111, and MT133; M02, MT112, and MT134; M05, MT113, and MT135; and M11, MT114, and MT136. Activities toward benzyloxyresorufin (10 µM) were determined by quantifying the fluorescent signals of the corresponding resorufin product peaks formed. BROD-induced NADPH consumption was determined by quantifying the decreases of the corresponding UV signals at 340 nm. (C) Corresponding BROD activities as measured by the cuvette assay are depicted. AUC, area under the curve; FLD, fluorescence detection; RES, resorufin; UV, UV detection.

CYP BM3 Organic Solvent Tolerability Screening in FIA Mode

After having established that the FIA EAD setup could be employed to accurately measure BROD activity and NADPH consumption in parallel, the assay was used to screen the activity of the CYP BM3 mutant library in the presence of different percentages of MeOH, ACN, and isopropanol (iPrOH). Figures 2 and 3 display the relative activities of the various mutants at different percentages of MeOH and ACN, respectively. Supplementary Figure S3 displays relative activities of the various mutants at different percentages of iPrOH. Corresponding amounts of resorufin formed and NADPH consumed are provided in the Supplementary Material.

Figure 2.

Relative activities as a function of methanol (MeOH) concentration as measured by flow-injection analysis into the enzyme activity detection setup. Relative activities at 1%, 5%, 10%, and 20% of MeOH are calculated as the ratio of the corresponding specific activities to the specific activity in the presence of 1% of MeOH.

Figure 3.

Relative activities as a function of acetonitrile (ACN) concentration as measured by flow-injection analysis into the enzyme activity detection setup. Relative activities at 1%, 5%, 10%, and 20% of ACN are calculated as the ratio of the corresponding specific activities to the specific activity in the presence of 1% of ACN.

As shown in Figure 2 , some of the novel mutants displayed increased tolerability toward MeOH. Although a downward trend for templates M02, M05, and M11 is seen in relative activity with an increasing percentage of MeOH, the corresponding triple mutants (MT112, MT113, and MT114, respectively) display an increase in relative activity at 5% and 10% of MeOH. The increase in tolerability toward MeOH is highest for MT112, the triple mutant of M02, which appeared to be active at 20% MeOH. For the triple mutants of M01, M02, and M05 (MT111, MT112, and MT113, respectively), the activity at 20% of MeOH is higher than that of their corresponding templates. This effect is not observed for the triple mutant of M11. Addition of the T235A mutation to mutants MT111, MT112, MT113, and MT114 (MT133, MT134, MT114, and MT136, respectively) appeared to reverse the tolerability to MeOH to a large extent ( Fig. 2 ).

Figure 3 shows that the triple mutants of M01, M02, and M05 also display an increased tolerability toward ACN compared with their templates. It is again observed that this effect is strongest for the triple mutant of M02. Overall, the negative effect on the activity of all tested CYP BM3 mutants caused by increasing the amount of ACN is much stronger compared with the effect of increasing the amount of MeOH. In the study by Wong et al.,¹¹ the effect of MeOH on CYP BM3 mutants was not tested. However, a stronger inhibition of ACN was observed compared with ethanol.

As shown in Supplementary Figure S3, increasing the concentration of iPrOH negatively affects the activity of all tested CYP BM3 mutants. However, only M112, the triple mutant of M02, displays an enhanced tolerability toward iPrOH compared with its template at both 2.5% and 5% of iPrOH.

When looking at the effects of increasing the amounts of organic modifier on BROD activities and NADPH consumption, it was observed that a change in activity is always accompanied by a similar change in NADPH consumption (see Supplementary Tables S4 to S9). These findings suggest that the mutations introduced may indeed affect the electron transfer and functionality of the CYP BM3 mutants through a change in the spatial orientation of the heme¹⁹ and the reductase domains as proposed by Wong et al.¹¹

Furthermore, consistent with the results of Wong et al., the effects of the stabilizing mutations appear to be dependent on the organic solvent used. However, the specific mechanisms underlying the solvent dependence of effects are still unknown.

CYP BM3 Organic Solvent Tolerability Screening in Gradient Mode

As described above, a number of mutants display an increased organic solvent tolerability toward MeOH, ACN, and iPrOH. Because this effect was most significant for MeOH, it was decided to investigate this in more detail. In the FIA EAD setup, the activity of the different mutants was tested at only four discrete MeOH concentrations. To construct activity curves at a large, continuous range of organic modifier concentrations, the setup was adapted to allow for measurement of parallel BROD activity and NADPH consumption against a gradient of an organic modifier. To test the applicability of the novel gradient EAD setup, the BROD activity and BROD-induced NADPH consumption of the 12 CYP BM3 mutants was screened against a gradient of MeOH. Instead of using a carrier solution with a fixed percentage of MeOH, the two pumps controlling the carrier flow were used to run a gradient of MeOH from 1% up to 49%.

Figure 4A displays BROD activity profiles of M02 and its triple (MT112) and quadruple (MT134) mutants. Figure 4B displays the corresponding BROD-induced NADPH consumption traces. Similar profiles were generated for the other nine CYP BM3 mutants (data not shown). Activity of each mutant was tested in duplicate (two gradient runs were performed) and the raw data files of gradient EAD screening of M02 and MT112 are displayed in Supplementary Figure S4. The averaged BROD activity trace generated by M02 ( Fig. 4A ) shows that as soon as the gradient starts after the initial equilibration phase at 1% of MeOH, a significant decrease in activity is observed. At 25% of MeOH, the BROD activity is completely lost as the fluorescent response has reached the background level. The corresponding averaged BROD-induced NADPH consumption trace shows that slightly more NADPH is consumed initially (detected as a decrease in the UV response at 340 nm). NADPH consumption reaches a maximum around 30% of MeOH and is followed by a significant decrease, thereby indicating that the enzyme is no longer active. The averaged BROD activity trace generated by MT112 shows that a significant enhancement in activity is observed when the percentage of MeOH is increased. Although the absolute BROD activity of MT112 at low percentages of MeOH is smaller than that of M02, the absolute BROD activity of MT112 exceeds that of M02 at MeOH percentages above approximately 8%. This is in excellent agreement with the findings of the FIA EAD screening experiment. Absolute BROD activity seems to reach a maximum at approximately 15% of MeOH and thereafter decreases. However, even at 25% of MeOH, MT112 still displays significant activity. The BROD-induced NADPH consumption trace also shows a significant enhancement when the percentage of MeOH is increased, which agrees very well with the FIA EAD results. This increase in NADPH consumption is much higher than the M02-mediated increase. From the BROD activity and BROD-induced NADPH consumption traces, it can be observed that MT134 displays no increased tolerability toward MeOH, which corresponds with the FIA EAD results. BROD activity and BROD-induced NADPH consumption traces were also generated for M01, M02, and M11 and their corresponding triple and quadruple mutants (data not shown).

Figure 4.

(A and B) Benzyloxyresorufin O-dealkylation (BROD) activity (A) and BROD-induced NADPH consumption activity traces (B) of M02, MT112, and MT134 measured in the enzyme activity detection setup in gradient mode. Traces depicted are averages of two individual measurements per mutant while methanol (MeOH) was used as an organic modifier. The dashed vertical lines indicate where the gradient was started (1) and where the gradient was finished (49).

BROD activity traces measured were subsequently used to calculate relative activities of the CYP BM3 mutants over a range of MeOH concentrations. Resulting activity curves are depicted in Figure 5 ; compared with their templates, the triple mutants of M01, M02, and M05 display an enhanced tolerability toward MeOH. This effect is greatest for MT112. When comparing results obtained for MeOH in the FIA EAD setup ( Fig. 2 ) with the results obtained for MeOH in the gradient EAD setup ( Fig. 5 ), it is clear that they correlate well. M02, M05, and M11 again display a downward trend in their relative activities with an increasing percentage of MeOH, whereas M01 shows a small initial increase followed by a decrease in relative activity. For M11 and its triple and quadruple mutants, it is again found that the introduction of the mutations has not resulted in an increase in tolerability toward MeOH. To confirm the results obtained in the two EAD setups, the BROD activity of M01, M02, M05, and M11 and their triple mutants was also tested in the traditional microplate reader-based assay format¹² at various concentrations of MeOH (data not shown). The results of this experiment confirmed that the triple mutants of M01, M02, and M05 displayed an enhanced tolerability toward MeOH, whereas this effect was not observed for the triple mutant of M11.

Figure 5.

Relative activities as a function of methanol (MeOH) concentration as measured in the enzyme activity detection setup in gradient mode. Relative activities at a given percentage of MeOH are calculated as the ratio of the corresponding specific activities to the specific activity in the presence of 1% of MeOH.

The results presented here clearly demonstrate that the novel gradient EAD setup is an ideal method to study organic solvent tolerability of CYP BM3 mutants. As a proof of principle, the effect of the organic modifier MeOH upon CYP-mediated BROD activity was determined. The setup of the assay can easily be adapted to test the effects of other organic modifiers, other CYP-mediated reactions that are suitable for an online screening assay, or other proteins that can be used in an online screening assay.

CYP BM3–Mediated Metabolism of the Drug Amitriptyline

The novel mutant that displayed the highest increase in organic solvent tolerability was MT112, the triple mutant of M02. To test whether MT112 also displayed an enhanced tolerability toward organic solvents while metabolizing substrates other than the drug-like alkoxyresorufins, an experiment was performed to investigate the M02- and MT112-mediated metabolism of the drug amitriptyline. Amitriptyline was previously shown to be efficiently metabolized by M02²⁰ in the presence of increasing concentrations of DMSO, because this is the most commonly used organic solvent for poorly soluble substrates. It was previously shown that the mutations applied in this study strongly increased tolerance of CYP BM3 F87A for DMSO.¹¹

Amitriptyline was metabolized to four products by both mutants (see the Supplementary Material for details). Three monohydroxylated products, (E)-10-OH-amitriptyline (OH1), (Z)-10-OH-amitriptyline (OH2), and OH3, and the demethylated product nortriptyline, were formed. For quantification of the parents and metabolites, the peak areas from the MS spectra were used assuming that they yielded similar MS responses, although it is realized that these responses may differ. However, UV data could not be used for quantification because not all peaks were baseline separated. The obtained results were used to calculate the amount of amitriptyline depletion by the two mutants at various DMSO concentrations. As shown in Figure 6A , the activity of M02 toward amitriptyline is higher than that of MT112 at all tested DMSO concentrations. However, from the relative activities of both mutants depicted in Figure 6B , it can be seen that MT112 displays an enhanced tolerability toward DMSO compared with M02. To illustrate that the metabolic profiles generated by the two mutants were similar, representative extracted ion chromatograms are included in Supplementary Figures S5 and S6. Metabolite distribution patterns generated by the two mutants at different percentages of DMSO are also included in Supplementary Figure S7. Metabolic profiles generated by the two mutants are very similar, because they both display a shift toward formation of nortriptyline and a decrease in formation of OH2 at higher DMSO percentages. The results from this experiment clearly show that MT112, the M02 triple mutant, displays enhanced organic solvent tolerability when amitriptyline is incubated in the presence of DMSO. Introduction of the R471A E494K S1024E triple mutation, however, results in a significant decrease in overall metabolic activity.

Figure 6.

Metabolism of amitriptyline by M02 and MT112. (A) Substrate depletion at a given percentage of DMSO is calculated by using the averaged peak area of the parent at 30 min and at time zero. Values represent the mean of two replicates and are expressed in percentages of the averaged peak area of the parent at time zero. Variability of duplicate experiments was below 5%. (B) Relative activities at a given percentage of DMSO are calculated as the ratio of the corresponding substrate depletions to the substrate depletion in the presence of 1% of DMSO.

In conclusion, the results of this study show that continuous-flow bioassays can be used to screen CYP BM3 mutants for their tolerability toward organic solvents, both at fixed concentrations of organic solvents and with gradients. The bioassay used in this study was based on the CYP BM3–mediated O-dealkylation of alkoxyresorufin to the highly fluorescent product resorufin. In principle, this setup will also be applicable for other fluorogenic substrates such as alkoxycoumarins, which have also been demonstrated to be converted to highly fluorescent products.^21–23 Alternatively, chromogenic substrates that are converted to colorimetric products, such as p-nitrophenol ethers, will be applicable for the online activity methodology.²⁴ The fact that the presented methodology can also be used to measure NADPH consumption shows that the setup is not limited to fluorogenic and chromogenic substrates. However, although NADPH oxidation has been applied as a generic method for measuring CYP activities,^25–27 it does not necessarily represent product formation because several substrate/CYP combinations show a high degree of uncoupling, as is the case with metabolism of alkoxyresorufins by CYP BM3 mutants. The most generic method therefore would be online detection of metabolites by MS, as has been successfully applied for analysis of products formed by CYP BM3–mediated metabolism of steroids and p38α kinase inhibitors.^28,29 However, this methodology requires availability of expensive mass spectrometers, which will be less commonly available for screening purposes than fluorometric or spectrophotometric detectors.

Footnotes

Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at .

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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