Catalytic enhancements in cytochrome P450 2C19 by cytochrome b₅

Abstract

Human cytochrome P450 2C19 catalyzes P450 enzyme reactions of various substrates, including steroids and clinical drugs. Recombinant P450 2C19 enzyme with histidine tag was successfully expressed in Escherichia coli and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mass (UPLC-MS/MS) spectrometry showed that the purified P450 2C19 enzyme catalyzed 5-hydroxylation reaction of omeprazole. The purified enzyme displayed typical type I binding spectra to progesterone with a K_d value of 4.5 ± 0.2 µM, indicating a tight substrate binding. P450 2C19 catalyzed the hydroxylation of progesterone to produce 21-hydroxy (OH) as a major and 17-OH product as a minor product. Steady-state kinetic analysis of progesterone 21-hydroxylation indicated that the addition of cytochrome b₅ stimulated a five-times catalytic turnover number of P450 2C19 with a k_cat value of 1.07 ± 0.08 min⁻¹. The molecular docking model of progesterone in the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme with a distance of 4.7 Å, suggesting 21-hydroxylation of progesterone is the optimal reaction of P450 2C19 enzyme for a productive orientation of the substrate. Our findings will help investigate the extent to which cytochrome b₅ affects the metabolism of P450 2C19 to drugs and steroids.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43188-023-00219-8.

Introduction

Cytochrome P450s (P450s) belong to the monooxygenase superfamily of heme-thiolate proteins [1, 2]. Human P450s enzymes catalyze the oxidation of various chemicals, including drugs and endogenous steroidal compounds [3]. The P450 2C subfamily is composed of P450 2C8, 2C9, 2C18, and 2C19, accounting for approximately 20% of the cytochrome P450 in the human liver [4–6]. P450 2C19 is clinically important because it metabolizes approximately 10% of marketed drugs [7]. The P450 2C19 gene is highly polymorphic, and some polymorphisms in it are known to affect drug efficacy and drug-drug interactions [8]. Omeprazole, a proton pump inhibitor in the stomach, is mainly metabolized by P450 2C19, and 5-hydroxylation of omeprazole has been used as a biomarker reaction for P450 2C19 (Fig. 1) [9, 10]. P450 2C19 also metabolizes endogenous compounds including steroid hormones, such as progesterone and testosterone [11]. This enzyme catalyzes progesterone oxidation to 21-hydroxyprogesterone as the major product (Fig. 1) [12]. Generally, 21-hydroxylation of progesterone and 17-hydroxyprogesterone is mainly catalyzed by P450 21A2 in the adrenal cortex, and steroid 21-hydroxylase deficiency (21-OHD) caused by a mutation in CYP21A2 gene severely impairs the synthesis of aldosterone and cortisol [13]. A previous study reported that progesterone 21-hydroxylation of a homozygous hepatic P450 2C19 ultra-metabolizer allele could contribute to the alleviation of 21-OHD, although 21-hydroxylation of progesterone by P450 2C19 is about one-fifth the catalytic efficiency of P450 21A2 [14].

Fig. 1.

Oxidation reactions by P450 2C19. P450 2C19 metabolizes omeprazole to produce 5-hydroxyomeprazole and progesterone to produce 21-hydroxyprogesterone

Cytochrome b₅ (CYB5A, b₅) is a heme protein involved in various electron transfer metabolic pathways [15]. It is also bound to the endoplasmic reticulum membrane in several tissues, including the liver [16]. The catalytic activities of P450 enzymes are affected by the b₅ protein; In particular, the strong enhancement of the P450 17A1 lyase reaction to C21 steroids (17-OH progesterone and 17-OH pregnenolone) has been intensively studied [17–19]. The b₅ protein stimulates P450 17A1 lyase activity through interaction with a ternary complex composed of P450: b₅: P450-NADPH reductase (POR) [17]. A previous study by Yamazaki et al. reported that the omeprazole 5-hydroxylation reaction of P450 2C19 was stimulated by b₅ and showed that this simulation of P450 2C19 activity did not require direct electron transfer in reconstituted systems [20].

In this study, we constructed a histidine-tagged P450 2C19 vector, purified the enzyme, and analyzed the stimulatory effect of cytochrome b₅ on the catalytic activity of P450 2C19 in the oxidation of omeprazole and progesterone.

Materials and methods

Chemicals

Omeprazole, progesterone, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP⁺, Formic acid and dilauroyl-_L-phosphatidylcholine (DLPC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 21-Hydroxyprogesterone was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Ni²⁺-nitrilotriacetate (NTA) agarose was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) was purchased from GoldBio (St. Louis, MO, USA). Rat NADPH-P450 reductase (NPR) was heterologously expressed in E. coli strain HMS174 and purified as described previously [21]. All chemicals were of the highest commercial grade and commercially available.

Construction of P450 2C19 with his-tag expression plasmids

The open reading frame (ORF) regions of P450 2C19 gene were amplified by polymerase chain reaction (PCR) with primers including NdeI and HindIII restriction sites and 6 × Histidine-tag at C-terminus; the primer sequences were 5’-CCCTACACATATGGCTCTGTTATTAGCAGT-3’, 5’-CAAAAGCTTTCAATGGTGGTGGTGATGGTGGACAGGAATGAAGCACAG-3’. The N-terminal modification of the MALLLAVF sequence originated from bovine P450 17A1 to maximize P450 expression (22). The amplified PCR-fragment was separated by agarose gel electrophoresis and cloned into the pCW Ori vector. The constructed expression plasmid of P450 2C19 with a histidine-tag was verified by restriction enzyme digestion and nucleotide sequencing.

Expression and purification of recombinant P450 2C19 enzyme

The expression and purification of the P450 2C19 enzyme were performed as previously described, with some modifications [21, 23]. E. coli DH5α cells were transformed with the constructed pCW expression vector. Transformed cells were spread on Luria–Bertani (LB) plate containing 50 μg/ml ampicillin and then incubated overnight at 37 °C. One colony was inoculated into 6 ml of LB medium containing 50 μg/ml ampicillin and grown overnight by shaking in an incubator at 37 °C and 230 rpm. The cultures were transferred to 500 ml of Terrific broth (TB) medium containing 50 μg/ml ampicillin and incubated at 37 °C and 230 rpm. Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), 1 mM thiamine, 0.5 mM 5-aminolevulinic acid and trace elements. The expression medium was incubated at 28 °C and 200 rpm for 48 h and harvested by centrifugation at 4000 rpm for 30 min. The harvested cells were resuspended in TES buffer containing 100 mM Tris–acetate, 0.5 mM EDTA, and 500 mM sucrose and incubated at 4 °C for 30 min with lysozyme and 100 mM phenyl-methylsulfonyl fluoride. The cells were centrifuged at 4000 rpm for 30 min and sonicated in sonication buffer containing 100 mM potassium phosphate (pH 7.4), 20% glycerol (v/v), and 0.1 mM dithiothreitol. The membrane fraction was centrifuged at 5,000 g for 20 min and the supernatant was centrifugated at 65,000 × g for 2 h. The supernatant was solubilized in solubilization buffer containing 100 mM potassium phosphate, 20% glycerol (v/v), 10 mM β-mercaptoethanol, 1 mM EDTA and 1% CHAPS (w/v) and then centrifuged at 65,000 × g for 2 h. The supernatant containing the solubilized proteins was loaded onto an NTA agarose column that was pre-equilibrated with 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.5 M NaCl, and 5 mM imidazole. The column was washed with 100 mM potassium phosphate (pH 7.4) containing 20% glycerol, 0.5 M NaCl and 20 mM imidazole. The protein fractions were eluted with 100 mM potassium phosphate (pH 7.4) containing 20% glycerol, 0.5 M NaCl and 300 mM imidazole. The eluted protein fraction was concentrated using an Amicon Ultra-30 centrifugal filter (Millipore, Billerica, MA, USA) and dialyzed with 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.1 mM 1,4-dithiothreitol at 4 °C.

Progesterone binding titration analysis

Purified P450 2C19 proteins were diluted to 2 μM with 100 mM potassium phosphate buffer (pH 7.4) and divided into two glass cuvettes. Spectroscopic changes (350–500 nm) were measured by the addition of ligands using a CARY 100 Varian spectrophotometer (Agilent Technologies, CA. USA). Substrate binding titration analysis was performed using progesterone. Substrate binding affinity (K_d) was calculated by plotting the difference between the maximum and minimum wavelengths of absorbance against the substrate concentration.

Omeprazole 5-hydroxylation assay

5-Hydroxylation of omeprazole was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) (Waters, Milford, MA, USA). The reaction mixture contained 10 pmol P450 2C19, 20 pmol rat NPR, 0–500 pmol cytochrome b₅, 10 μM omeprazole, and 30 μg 1,2-dilauroyl-sn-glycerol-3-phosphocholine (DLPC) in 500 μL of 100 mM potassium phosphate buffer (pH 7.4). After 3 min of preincubation at 37 °C, the reaction was initiated by adding an NADPH-generating system (10 mM NADP⁺, 100 mM glucose 6-phosphate, and glucose-6-phosphate dehydrogenase). The reaction proceeded for 10 min at 37 °C and was terminated by adding 1 mL of CH₂Cl_2. The samples were vortexed and centrifuged at 3,500 rpm for 15 min. The layer of CH₂Cl₂ was extracted into a test tube and dried under N₂ gas. The dried samples were dissolved in 200 µl of CH₃CN. The samples were injected into an ACQUITY UPLC™ BEH C18 column (50 × 2.1 mm, 1.7 µM) equipped with Waters ACQUITY UPLC™ (Waters, Milford, MA) and Waters Quattro Premier™ (Waters, Milford, MA). For analysis of the reaction samples, the mobile phase comprised H₂O containing 10% CH₃CN with 0.1% formic acid (A) and 100% CH₃CN with 0.1% formic acid (B) at a flow of 0.3 mL/min. The mobile phase B was maintained at 10% for 0.5 min and increased to 50% for 2.5 min and maintained at 50% for 0.5 min and subsequently decreased to 10% for 1.5 min. The mobile phase B was maintained at 10% for 0.5 min. Positive electrospray ionization and multiple reaction monitoring (MRM) modes were used for the analysis [omeprazole (m/z 346.1 > 197.8) and 5-hydroxyomeprazole (m/z 362.1 > 152.1)]. The injection volume was 3 μl. The capillary voltage was 3.5 kV and the cone voltage was 25 V. The column temperature was maintained at 40 °C. The source’s temperature was maintained at 150 °C. The desolvation temperature was maintained at 500 °C. The desolvation gas flow rate was set at 550 l/h. The cone gas flow rate was set at 50 l/h. The metabolic products were integrated. Steady-state kinetic parameters were calculated using GraphPad Prism software (GraphPad, Inc., La Jolla, CA, USA).

Progesterone 21-hydroxylation assay

Progesterone hydroxylation was analyzed using UPLC-MS/MS. The reaction mixture contained 50 pmol P450 2C19, 100 pmol rat NPR, 200 pmol b₅ (or no b₅), and 30 μg DLPC in 500 μL of 100 mM potassium phosphate buffer (pH 7.4). The same procedures was used for the P450 reactions. The extracted samples were then injected into UPLC-MS/MS system. The UPLC mobile phase consisted of H₂O containing 10% CH₃CN with 0.1% formic acid (A) and 100% CH₃CN with 0.1% formic acid (B) at a flow of 0.2 mL/min. The mobile phase B was maintained at 5% for 0.5 min and increased to 40% for 2.5 min and maintained at 40% for 0.5 min, and then increased to 90% for 1 min. The mobile phase B maintained at 90% for 0.5 min and decreased to 5% for 1 min, and then maintained at 0.5 min. Positive electrospray ionization and a MRM modes were used to analyze the reaction [progesterone (m/z 315.2 > 109.1), and 21-hydroxy progesterone (m/z 331.2 > 109.1)]. The metabolic products were integrated. Steady-state kinetic parameters were calculated using GraphPad Prism software (GraphPad, Inc., La Jolla, CA, USA).

Molecular docking modeling of P450 2C19

A docking modeling analysis of the P450 2C19 complex with progesterone was performed using Autodock 4.2 software (The Scripps Research Institute, La Jolla, CA, USA).

The structure of P450 2C19 was obtained from the Protein Data Bank (PDB ID: 4GQS). Before docking modeling, all water molecules and ligands except for the prosthetic heme groups were removed from PDB.

Results

Expression and purification of P450 2C19

P450 2C19 protein with a 6 × histidine tag was expressed in E. coli DH5α cells. CO binding difference spectra of the reduced P450 enzymes in whole cells confirmed the expression of P450 2C19 (Fig. 2A). The maximum expression of P450 2C19 was observed at 230 nmol/liter culture at 48 h. The membrane fractions were solubilized, and the P450 protein in the soluble fractions was purified using Ni²⁺-NTA affinity column chromatography. Spectral analysis of purified P450 2C19 revealed a typical CO binding Soret peak at 450 nm (Fig. 2B). The absolute spectra of ferric forms indicated a low spin state with Soret bands at 419 nm and smaller α-bands and β-bands at 571 nm and 538 nm (Supplementary Fig. S1). The ferrous form of P450 2C19 reduced by sodium dithionite showed a broad peak around 413 nm and shifts of α-bands and β-bands to 560 nm and 539 nm (Supplementary Fig. S1).

Fig. 2.

Expression and purification of P450 2C19 enzyme. a CO binding spectra of P450 2C19 at the E. coli whole-cell. b CO binding spectra of purified P450 2C19

Binding analysis of P450 2C19 to progesterone

Spectral binding titration analysis of P450 2C19 was performed using progesterone. The binding analysis of P450 2C19 with progesterone showed a specifically shifted type I spectral change, with an increase at 412 nm and a decrease at 432 nm (Fig. 3), indicating that progesterone occupies the distal site of the low-spin hexa-coordinated P450 heme. The K_d value for progesterone was 4.5 ± 0.2 µM, indicating a tight substrate binding affinity of progesterone to P450 2C19.

Fig. 3.

Binding analysis of P450 2C19 to progesterone. P450 2C19 binding titration with progesterone. The insets show the plots of Δ(Abs_max – Abs_min) vs. the concentration of substrate

Omeprazole hydroxylation and stimulation by cytochrome b₅

Omeprazole hydroxylation by P450 2C19 was analyzed using UPLC-MS/MS. P450 2C19 hydroxylates omeprazole to 5-hydroxyomeprazole. The reaction product, 5-hydroxyomeprazole, was detected in MRM mode at m/z 362.1 > 152.1 (Supplementary Fig. S2). The peak produced in the P450 2C19 reaction was confirmed by co-elution of chromatography with an authentic 5-hydroxyomeprazole compound. To analyze the stimulatory effect of b₅, the catalytic activity of P450 2C19 was analyzed at various ratios of b₅. As shown in Fig. 4A, omeprazole hydroxylation of P450 2C19 was stimulated by b₅ in a concentration-dependent manner (Fig. 4A). Steady-state kinetic analysis of the reaction of P450 2C19 with a 20-fold concentration of b₅ significantly increased catalytic efficiency (k_cat/K_m, ~ 61%), mainly owing to an increase in k_cat value (Fig. 4B, Table 1).

Fig. 4.

Omeprazole hydroxylation by P450 2C19 and stimulatory effect of b₅. A Omeprazole hydroxylation activities of P450 2C19 were analyzed with 0 to 50 ratios of b₅. Each bar represents the SD of triplicate assays B The steady state kinetic analysis in the reaction of P450 2C19 without b₅ and with 20-fold concentration of b₅. Each point represents the mean ± SD of triplicate assays. Steady-state kinetic parameters are shown in Table 1

Table 1.

Steady-state kinetic parameters of P450 2C19 and stimulation effect by b₅

5-Hydroxylation of omeprazole				21-Hydroxylation of progesterone
P450: b5 ratio	kcat (min−1)	Km (μM)	kcat/Km	P450: b5 ratio	kcat (min−1)	Km (μM)	kcat/Km
1: 0	4.2 ± 0.2	8.5 ± 1.0	0.49 ± 0.06	1: 0	0.22 ± 0.01	11.0 ± 0.8	0.020 ± 0.002
1: 20	7.2 ± 0.4	9.1 ± 1.6	0.79 ± 0.15	1: 4	1.07 ± 0.08	14.7 ± 2.6	0.073 ± 0.014

Progesterone hydroxylation and stimulation by cytochrome b₅

The progesterone 21-hydroxylation activity of P450 2C19 has been previously reported [12]. In this study, the catalytic activity of P450 2C19 in progesterone hydroxylation was analyzed using UPLC-MS/MS. P450 2C19 catalyzes the hydroxylation of progesterone, yielding a 21-hydroxy product (eluted at 2.2 min) as the major product and 17-hydroxyprogesterone (eluted at 2.5 min) as the minor product (Supplementary Fig. S3). The 21-hydroxylated progesterone was analyzed using the MRM mode of m/z 331.2 > 109.1 (Supplementary Fig. S3). The reaction product was confirmed by co-elution of chromatography using authentic 21-hydroxyprogesterone. Steady-state kinetic analysis of progesterone 21-hydroxylation was performed and the stimulatory effect of b₅ was studied. The catalytic activity of progesterone 21-hydroxylation was markedly enhanced when b₅ protein (with a 1:4 ratio of P450: b₅) was added to the reactions (Fig. 5 and Table 1). The catalytic efficiency (k_cat/K_m) and k_cat value were stimulated approximately 4 and fivefold, respectively, when b₅ was added (Fig. 5 and Table 1). These results indicated that the stimulatory effect was more dramatic in the progesterone 21-hydroxylation reaction.

Fig. 5.

Steady-state kinetic analysis of progesterone hydroxylation by P450 2C19 and stimulatory effect of b₅. Steady-state kinetic analysis of progesterone 21-hydroxylation by P450 2C19 without b₅ and with a fourfold concentration of b₅. Each point represents the mean ± SD of triplicate assays. Steady-state kinetic parameters are shown in Table 1

Docking model of P450 2C19 with progesterone

To study the function-structure relationship of progesterone in P450 2C19, molecular docking modeling was performed. The reported X-ray crystal structure of P450 2C19 (PDB ID code: 4GQS) was used for docking. Based on the RMSD and docking energy, the best docking model was selected. The molecular model of the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme in the active site of P450 2C19 with 4.7 Å (Fig. 6A). This docking model suggested that 21-hydroxylation of progesterone was the optimal the reaction of P450 2C19 enzyme for productive orientation of the substrate.

Fig. 6.

Molecular docking modeling of P450 2C19 with progesterone. A docking modeling of the P450 2C19 with progesterone was conducted with the Autodock 4.2 software. Progesterone is colored with cyan and heme is colored with red. I-, F-, and G-helices was colored in green. a In this docking model, carbon 21-carbon of progesterone was located 4.7 Å from the heme iron atom of P450 2C19. b Asn204 of the F helix forms a hydrogen bond with the carbonyl group (O3) of progesterone with a distance of 3.7 Å. Progesterone forms hydrophobic interaction with Leu237, Gly296, and Leu366 in the active site of P450 2C19

Discussion

In this study, a recombinant P450 2C19 enzyme with a 6 × histidine tag modification at the C-terminus was purified, and its ability to catalyze 5-hydroxylation of omeprazole and 21-hydroxylation of progesterone was analyzed. Binding analysis of P450 2C19 to progesterone showed shifted type I spectral changes (Fig. 3). Interestingly, the binding analysis of P450 2C19 to Δ5-steroid (pregnenolone) did not show any spectral change (data not shown), suggesting that the endogenous catalytic activity of P450 2C19 is specific to Δ4-steroid (progesterone).

Omeprazole is a well-known proton pump inhibitor and a biomarker drug for studying the metabolism of P450 2C19. A previous study reported that P450 2C19 is influenced by cytochrome b₅ through an allosteric effect without direct electron transfer [24]. In this study, the activity of P450 2C19 increased in a concentration-dependent manner with cytochrome b₅, and a maximal increase was observed upon in the addition of 20-fold b₅ (Fig. 4). Although this stimulatory effect of cytochrome b₅ is not essential for P450 2C19 metabolism, mutations in the interaction surface of P450 2C19 and b₅ can affect the metabolic efficiency of the enzyme [24]. In a previous study, the effect of b₅ on the drug metabolism ofP450 2C9, a member of the cytochrome P450 2C subfamily, was investigated, and the maximum rate of catalysis was observed when the ratio of b₅ was fourfold that of P450 but this enhancement decreased when excess b₅ was added [25]. Conversely, our result showed that omeprazole 5-hydroxylation by P450 2C19 increased as the concentration of cytochrome b₅ increased (Fig. 4). This result suggests that the stimulatory effect of b₅ may differ among various P450 enzymes.

P450 2C19 metabolizes steroid hormones, including progesterone, and produces 21-hydroxyprogesterone by hydroxylating the 21-carbon position of progesterone [12]. The expression levels of cytochrome b₅ in human liver microsomes showed high variation, and there was no significant correlation between the relative content of b₅ and omeprazole 5-hydroxylation by P450 2C19 in human liver microsomes. [26]. In our study, we investigated the kinetics of 21-hydroxyprogesterone produced by P450 2C19 in the b₅ and then compared it with that of no cytochrome b₅ (Fig. 5). After the reaction of P450 2C19 with progesterone, we quantified 21-hydroxyprogesterone using UPLC-MS/MS after the extraction process. In the presence of cytochrome b₅, the catalytic efficiency (k_cat/K_m) for progesterone 21-hydroxylation was 0.020 ± 0.002. When the ratio of cytochrome b₅ to P450 2C19 was 4, the catalytic efficiency of progesterone 21-hydroxylation was 0.073 ± 0.014, approximately fourfold higher than when cytochrome b₅ was not added (Table 1). In metabolizing the endogenous compound, b₅ stimulated the reaction and the metabolism of the progesterone by P450 2C19. This result suggests that the stimulatory role of b₅ may affect the pharmacokinetic fates of P450 2C19-catalyzing drugs and steroids. Previous studies reported some correlation between b₅ affinity and stimulation of catalytic activities of human P450 enzymes [20, 27]. Catalytic activities of P450 2A6, 2B6, 2C8, 2C9, 2E1, 4A11, 3A4, and 17A1 were enhanced by adding b5 [20, 27]. However, in case of P450 2S1, b₅ bound tightly to P450 enzyme but any stimulatory effect of b₅ on catalytic activity was not observed [27, 28].

P450 2C19 hydroxylates progesterone to produce 21-hydroxyprogesterone as a major product and 17α-hydroxyprogesterone, 16α-hydroxyprogesterone, and 6β-hydroxyprogesterone as minor products [12]. P450 21A2, which is mainly expressed in the adrenal cortex, metabolizes progesterone to 21-hydroxyprogesterone, which is necessary for aldosterone biosynthesis [29, 30]. In this study, docking modeling was performed with the Autodock Vina software program to confirm that the progesterone 21-hydroxylation of P450 2C19 was the major reaction. The best docking model was for C21 of progesterone to face Fe³⁺ in the heme prosthetic group. The docking energy was -11.2 kcal/mol. In this docking model, the distance between heme and C21 position of progesterone was 4.7 Å, sufficient for interaction between P450 2C19 and progesterone (Fig. 6A). The carbonyl group (O3) of progesterone could interact with Asn204 in F helix at a distance of 3.7 Å, forming a hydrogen bond. Progesterone bound to the active site of P450 2C19 through hydrophobic interactions with Leu237, Gly296, and Leu366 (Fig. 6B).

Supplementary Information

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Funding

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Data availability

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Declarations

Conflict of interest

The authors declare that they have no conflict of interest with the contents of this article.