An integrated screening system for the selection of exemplary substrates for natural and engineered cytochrome P450s

Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.

was confirmed by SDS-PAGE. The amount of functional P450 was calculated using the extinction coefficient of 91 mM -1 cm -1 at 450 nm. 3
After being stirred for 10 min at room temperature, then (-)-S3 (1.23 M, 1 mL, 1.23 mmol) was added. After being stirred at the same temperature for 3 h, the reaction was quenched with saturated aqueous NaHCO3 (2 mL) and the mixture was extracted with Et2O twice. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. Purification of the residue by flash chromatography (Et2O: Hexane = 1:16) gave 104 mg (0.321 mmol, 26%) of (-)-S5 as brown oil.

Synthesis of methyl xanthate (-)-S7
To a flame dried flask was added NaH (60%, 6.00 mg, 145 µmol) and THF (0.5 mL). The mixture was cooled to 0 °C and a solution of (-)-#104ox1 (1.76 mg, 7.26 µmol) in THF (2 mL) was added dropwise. The reaction mixture was stirred for 5 min at 0 °C, then allowed to warm to room temperature over 1 h. CS2 (10.0 µL, 145 µmol) was added to the reaction mixture at the same temperature and stirring was continued for an another 1 h. Then, MeI (20.0 µL, 321 µmol) was added to the mixture and stirring was continued for an additional 30 min. The reaction mixture was partitioned between 0.1 N HCl (1 mL) and Et2O (3 mL), and the aqueous layer was extracted with Et2O (10 mL × 3) and washed with brine (10 mL).
The reaction was quenched with sat. aq. NaHCO3 (0.2 mL) and extracted with AcOEt (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure.

Structure estimation of oxidized products by GC/MS
Each hit compound (20 µM) was incubated with P450 BM3(WT) or P450 BM3(F87A) (0.75 ~ 3.75 µM) in reaction buffer (100 mM KPi, 230 µM NADPH, 5% glycerol, pH 7.4, 5 mL) at 25 °C with shaking at 110 rpm. After the incubation for 0 min, 1 min, 10 min, 2 h, and 12 h, the reaction mixture was extracted with AcOEt (500 µL×3). For the reaction of the carboxylic acid substrate, 0.1 N HCl (50 µL) was added to the reaction mixture before extraction. The combined organic layers were dried down in vacuo. Pyridine (20 µL) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (30 µL) were added to the residue, and the resulting mixture was incubated at 60 °C for 1 h. The reaction mixture was diluted with CH3CN (50 µL), and analyzed by GC/MS using JMS-T100GCV time-of-flight mass spectrometer (JEOL, Tokyo, Japan) equipped with 7890A GC (Agilent Technologies, Santa Clara, CA, USA) and 7693 autosampler (Agilent) as follows: Five µL of each sample was injected with splitless mode at 250 ºC and analyzed by using HP-5 19091J-413 column (30 m × 0.25 mm id, film thickness 0.25 µm; Agilent Technologies). The carrier gas was helium at a constant flow-rate of 1.5 ml/min. The GC oven temperature was kept at 70 ºC for 4 min, ramped linearly from 70 ºC to 325 ºC at 30 ºC/min, and kept at 325 ºC for 7.5 min. The outlet of the column is directly connected to an electron ionization (EI, 70 eV) / field ionization (FI) combination source of the mass spectrometer and both EI and FI mode spectra were recorded in separate runs. Typical full width at half maximum mass resolution was around 8,000 and typical mass measurement accuracy was 5 mDa after application of single-point drift compensation with a column background peak at m/z 207 in EI mode.
The GC/MS data analysis was carried out in the following steps. First, the peaks that would be attributable to the monooxidized products, i.e., peaks of hydroxyl derivatives (detected as TMS-ether or TMS-ester) for each substrate were identified by comparing accurate mass chromatograms of the time-course samples. Typically, relative intensity of the peaks corresponding to the oxidized products increased with incubation time; some of the peaks are then decreased due to further oxidation and/or decomposition. Peaks stay the same in the time course were regarded as background and ignored.