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Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 from Streptomyces Species
1Department of Life Science and Biochemical Engineering, Sunmoon University, Asan 31460, Republic of Korea
2Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang-eup, Chungbuk 28116, Republic of Korea
3Genome-Based BioIT Convergence Institute, Asan 31460, Republic of Korea
4Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asan 31460, Republic of Korea
J. Microbiol. Biotechnol. 2021; 31(3): 464-474
Published March 28, 2021 https://doi.org/10.4014/jmb.2010.10020
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
The cytochrome P450s (CYPs) are a vast group of heme-containing enzymes found in virtually all living organisms. CYPs have a broad range of substrate specificity and are responsible for the hydroxylation of non-activated carbon atoms, dealkylation, epoxidation, demethylation, sulfoxidation, and carbon-carbon bond cleavage, making them an attractive target as biocatalysts for organic synthesis [1-5]. For catalysis, the majority of the microbial CYPs rely on one or more redox partners to supply redox equivalents. Cytochrome P450 reductases (CPRs) fused to a CYP domain as in the CYP102A and CYP116B family directly deliver electrons to the heme iron while NAD(P)H-dependent ferredoxin reductase (FDR) and ferredoxin (FDX) sequentially deliver electrons to the heme iron from the cofactor [6]. In addition, certain CYPs such as peroxygenases from the CYP152 family use hydrogen peroxide (H2O2) effectively, without the need for redox partners [7, 8]. Furthermore, some studies have used oxygen surrogates (OSs) to support CYP activity, although lower activity and rapid inactivation of the CYPs limited the efficiency of the OS-supported reactions [9, 10].
Steroids are ubiquitously available bioactive compounds with a wide range of therapeutic effects such as anti-inflammatory, immunosuppressive, anabolic, and diuretic activities [11-14]. Variations in the functional group, type, number, and attachment position to the core steroid subunit directly influence the biological function of steroids. Since steroids are hydrophobic, the presence of the hydroxyl group increases the polarity of the steroid. This may provide a higher level of biological activity or serve as an intermediate for the synthesis of steroidal derivatives. The presence of the hydroxyl group at the C7α position of dehydroepiandrosterone and pregnenolone displayed immunoprotective and immunoregulatory properties. Moreover, it was reported that the C11α-hydroxylation of deoxycortisone was crucial for anti-inflammatory activity, the 17β-hydroxyl function determined androgenic properties, and the 14β-hydroxyl group of steroids was cardio-active [15-19].
In eukaryotes and prokaryotes, steroid hydroxylation is accomplished exclusively by cytochrome P450 monooxygenases [20]. Bacterial monooxygenases compared to those in eukaryotes are advantageous for biotechnological applications because of their solubility and high level of expression [21]. A few CYPs are involved in the hydroxylation of steroids, like the CYP106A family (CYP106A1 and CYP106A2), the CYP109 family (CYP109B1 and CYP109E1), the CYP154C family (CYP154C2, CYP154C3, CYP154C4, CYP154C5, and CYP154C8), and CYP260A1 [17,22-27].
In this study, the cloning, heterologous overexpression, purification, and characterization of two CYP154C3s from
Materials and Methods
Materials
The steroids used in this study were obtained from TCI (Tokyo Chemical Industry Co., Ltd, Japan). Isopropyl-1-thio-β-D-galactopyranoside (IPTG), 1,4-dithiothreitol (DTT), and kanamycin were purchased from Duchefa Bohemie (Korea). Ampicillin, δ-aminolevulinic acid (ALA), NADH, and formate dehydrogenase were purchased from Sigma-Aldrich (Korea). All of the restriction enzymes, DNA polymerase, T4 DNA ligase, and dNTPs were purchased from Takara Bio (Japan). All other high-grade chemical products were obtained from commercially available sources.
Bioinformatics Analysis
The nucleotide sequences of CYP154C3-1 and CYP154C3‐2 were deposited in GenBank under accession numbers MF467273 and MT921810, respectively. Identification of the close homologs and comparison of the protein sequences were performed using the Basic Local Alignment Search Tool (BLAST). Multiple sequence alignment was accomplished using GeneDoc [28]. An evolutionary study was conducted using molecular evolutionary genetics analysis (MEGA X) [29]. A phylogenetic tree was constructed using the maximum likelihood method with 1000 bootstrap replicates and the evolutionary distances were computed using the Poisson correction method [30]. The CYP names were assigned by Dr. David Nelson (http://drnelson.utmem.edu/CytochromeP450.html).
Strains, Media, and Conditions
Molecular Cloning and Protein Over-Expression
The CYP154C3-1 and CYP154C3-2 encoding sequences (1,239 bp and 1,254 bp, respectively) were amplified from the genomic DNA of
For the in vitro reconstituted system, redox partners putidaredoxin (Pdx) and putidaredoxin reductase (PdR) were over-expressed and His-tagged in
Protein Purification and Concentration Determination
The crude extracts obtained by ultra-sonication were centrifuged at 12,500 rpm for 25 min at 4°C to remove cellular debris. The soluble fraction of the cell extracts was mixed with TALON His-tag resin pre-equilibrated with equilibrium buffer (potassium phosphate buffer pH 7.4) and shaken for 60 min. The protein-bound resin was eluted with elution buffers (potassium phosphate buffer pH 7.4 with 10% glycerol) containing 20 mM, 100 mM, and 250 mM imidazole. The fractions containing the proteins of interest were concentrated by ultrafiltration using Amicon centrifugal filters (Millipore) with a 30 kDa cutoff for CYP154C3s and PdR, whereas a 10 kDa cutoff was used for Pdx.
The concentration of the CYP154C3s was measured based on the CO-difference spectra method [32]. Using the potassium phosphate buffer protein, the protein was diluted to 2 ml and separated into two cuvettes (reference and sample). The sample cuvette was bubbled gently with carbon monoxide at a rate of 1 bubble per second for 1 min. Both the reference and sample were reduced with a few grains of sodium dithionite and the spectrum was recorded using a Biochrome Libra S35PC UV/Vis spectrophotometer (England). The concentration of functional CYP154C3s was estimated using an extinction coefficient ε450-490 of 91 mm-1 cm-1 [33]. The PdR concentration was estimated by calculating the average concentration from the 378 nm, 454 nm, and 480 nm wavelengths using extinction coefficients (ε) of 9.7, 10.0, and 8.5 mM-1cm-1, respectively [34]. The Pdx concentration was estimated by calculating the average concentration from the 415 nm and 454 nm wavelengths using extinction coefficients (ε) of 11.1 and 10.4 mM-1cm-1, respectively [31].
Effect of pH, Temperature, and Ionic Strength on Enzymatic Activity
The optimal pH for purified CYP154C3 activity was determined at 30°C using 50 mM potassium phosphate buffer with various pH ranges from 6.0 to 8.5. The maximal enzymatic activity at 30°C using phosphate buffer at pH 7.4 was defined as 100%. For the optimal temperature determination, the enzymatic activity assay was performed at various temperatures from 15–50°C in 50 mM phosphate buffer (pH 7.4). The maximum activity at 30°C in phosphate buffer (pH 7.4) was defined as 100%. The effect of the ionic strength maintained by sodium chloride (NaCl) on CYP154C3 activity was determined by adding different concentrations of NaCl (0–200 mM) into the reaction system. The maximal activity at 30°C in phosphate buffer (pH 7.4) with 70 mM NaCl was defined as 100%. Progesterone was used as the substrate for the characterization of both CYP154C3s in a reaction mixture containing 3 μM CYP154C3s, 100 μM substrate, 6 μM PdR, 24 μM Pdx, 100 μg/ml catalase, and 250 μM NADH. The reaction mixture was extracted twice with ethyl acetate and dried under vacuum for further analysis.
Enzymatic Activity Assay
The in vitro assay was carried out in the presence of NADH, H2O2, and PIDA. Ten substrates were used for the enzymatic activity assays (Fig. 1). The substrates were prepared by dissolving them in dimethyl sulfoxide (DMSO). The reaction mixture contained 3 μM CYP154C3s, 200 μM substrate, 6 μM PdR, 24 μM Pdx, 100 μg/ml catalase, and an NADH-regeneration system comprised of 1 U formate dehydrogenase, 150 mM sodium formate, and 1 mM MgCl2 in a final volume of 250 μl in 50 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of 250 μM NADH and incubated at 30°C for 2 h. The reaction mixture was extracted twice with 250 μl of ethyl acetate dried and analyzed by high-performance liquid chromatography (HPLC). Oxidizing agent (H2O2 and PIDA)-mediated conversion assays were also performed after determining the optimum concentration required for enzymatic activity. The reaction system consisted of 3 μM CYP154C3s and 200-μM substrate and was initiated by the addition of optimum concentrations of either H2O2 or PIDA. The reaction was incubated at 30°C for 2 h and extracted as described above.
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Fig. 1. Structure of the steroids used to determine the substrate specificity of the CYP154C3s.
Determination of Kinetic Parameters and Catalytic Efficiency
A reaction curve was generated for both the CYP143C3s by measuring the product formed over time from the different substrates. Product quantification was achieved by correlating the peak area of the product(s) with the total peak area of the product(s) and the substrate. The reaction mixture typically contained 1 μM CYP154C3s, 2 μM PdR, 8 μM Pdx, and 100 μM substrate. The reactions were initiated by adding 350 μM NADH. After establishing the initial velocity conditions, the concentrations of all substrates were varied in the range of 0–500 μM to generate a saturation curve. The
NADH-Coupling Efficiency
NADH oxidation rates were measured spectrophotometrically using purified CYP, PdR, and Pdx by monitoring the NADH absorbance at 340 nm over time. The reaction mixture typically contained 1 μM CYPs, 2 μM PdR, 10 μM Pdx, and 200 μM substrate in 50 mM phosphate buffer at pH 7.4. The reactions were initiated by the addition of 250 μM NADH (ε = 6.22 mM-1 cm-1) [31]. The substrate consumption was determined by HPLC. The background NADH consumption in the absence of substrate was also determined. The coupling efficiency was calculated as the percentage of NADH used for product formation over the total NADH consumption.
Determination of the Substrate Dissociation Constant
Substrate binding assays were performed by the spectrophotometric titration of the enzymes (CYP154C3s) in 50 mM potassium phosphate buffer (pH 7.4) with increasing substrate concentrations until saturation. The absorbance spectra of all samples were recorded from 350 to 500 nm using a Biochrome Libra S35PC UV/Vis Spectrophotometer (England). The
In Eq. (1), Aobs is the absorption shift determined at any ligand concentration, Amax is the maximal absorption shift obtained at ligand saturation, [Et] is the enzyme concentration used, [S] is the substrate concentration, and KD is the apparent dissociation constant for the enzyme–ligand complex.
HPLC and LC-MS Analysis
After drying, the extracted reaction mixture was used for analysis. The dried residue was dissolved in HPLC-grade methanol, filtered through a 0.45‐μm pore polytetrafluoroethylene filter, and analyzed by ultra-high-performance liquid chromatography (UHPLC). The sample was injected and separated using a Mightysil reverse phase C18 column (4.6 × 250 mm, 5 μm). Acetonitrile (B) and water (A) were used as the mobile phase in a gradient system of B at 15% for 0–10 min, 50% for 10–20 min, 70% for 20–25 min, and 15% for 25–40 min at a flow rate of 1 ml/min. Detection of the substrates and their product was performed by UV absorbance at 242 and 245 nm. Liquid chromatography-mass spectroscopy (LC-MS) analysis was performed with a SYNAPT G2-S/ACUITY UPLC liquid chromatography quadrupole time-of-flight/electrospray ionization mass spectrometer (Waters, USA) in the positive ion mode. The products formed were identified by comparison to products reported previously [17, 25, 36].
Results and Discussion
Bioinformatics Analysis
Multiple sequence alignment of the selected proteins was performed to observe sequence conservation and the presence of the signature motif. The characteristic conserved oxygen-binding and activation I-helix motif, K helix (EXXR) motif, and heme-binding domain for the CYP family were observed (Fig. 2). All proteins contained an acid-alcohol pair, glutamate, and a threonine residue, which facilitates oxygen activation in CYPs [37]. Homologs of the protein sequences were searched by conducting a PSI-BLAST search (NCBI server). A phylogenetic tree was constructed using the protein sequences of the CYP154C3s and their closest homologs (Fig. 3). Phylogenetic analysis revealed that the CYP154C3s clustered closer to the previously studied
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Fig. 2.
Multiple sequence alignment of The conserved and similar residues are highlighted. The highly conserved, functionally relevant regions (oxygen binding motif, EXXR motif, and heme‐binding signature motif) are underlined. The conserved cysteine residue of heme-binding motif is highlighted in red.Streptomyces CYP154Cs with CYP154C3, CYP154C4, and CYP154C5.
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Fig. 3.
Phylogenetic tree of The phylogenetic tree was based on amino acid sequences of the CYP154 family. The phylogenetic tree was constructed by the maximum likelihood method and the Poisson correction model. The prefixes in front of the CYP number abbreviate the genus and the species of the respective bacteria from where the enzyme originated (Faln,Streptomyces CYP154C3s and their closest homologs.Frankia alni ; Nfar,Nocardia Farcinia ; Save,Streptomyces avermitilis ; Sbin,Streptomyces bingchengensis ; Scar,Streptomyces carzinostaticus ; Scoe,Streptomyces coelicolor ; Sfra,Streptomyces fradiae ; Sgri,Streptomyces griseus ; Speu,Streptomyces peucetius ; Sroc,Streptomyces rochei ; Ssca,Streptomyces scabies ; Sspp,Streptomyces sp.; Stro,Salinispora tropica ; and Tfus,Thermobifida fusca ). The P450s in the current study are indicated by the symbol (◈). The vertical bar in the tree represents 0.2 amino acid substitutions per amino acid for the branch length.
Cloning, Overexpression, Purification, and Spectral Characterization of Proteins
The DNA fragments encoding the CYP154C3s genes were PCR-amplified and cloned into the pET32a (+) expression vector. Both CYPs, CYP154C3‐1 and CYP154C3‐2, showed a better co-expression of target proteins in the soluble form in
The cytosolic purified fraction of the CYP154C3s showed spectral properties characteristic of CYP enzymes by UV–Vis absorption spectroscopy. The carbon monoxide-bound, dithionite-reduced form of the CYP154C3s exhibited absorption maxima at 449 nm, the characteristic signature of CYP heme in its Fe2+CO complex form [38].
Substrate-Binding Assay
The binding of the substrates to the active site of CYP was observed by the displacement of the heme water ligand (the sixth ligand to heme iron). This resulted in a shift of the ferric heme iron from a low-spin to a high-spin state (so-called type I shift) with a minimum Soret absorption of around 420 nm and a maximum of around 390 nm [39, 40]. All of the steroids were tested for a possible type I spin shift in the CYP154C3s. Upon binding to CYP154C3s, the steroids exhibited a type I shift with a maximum absorbance at 390 nm and a minimum at 420 nm (Fig. S1). By titrating different concentrations of the substrates until saturation and fitting to a nonlinear tight-binding quadratic equation, the dissociation constant (
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Table 1 . The dissociation constant (
K d) of the CYP154C3s for steroids.SN Substrate K d [μM]CYP154C3-1 CYP154C3-2 1 Adrenosterone 0.318 ± 0.026 0.443 ± 0.035 2 Androstenedione 0.351 ± 0.033 0.475 ± 0.054 3 Corticosterone 14.060 ± 2.570 44.490 ± 3.970 4 Cortisone 50.730 ± 6.140 26.880 ± 3.510 5 Hydrocortisone 80.390 ± 15.850 107.280 ± 13.860 6 Nandrolone 13.530 ± 2.620 6.580 ± 0.840 7 Prednisolone 45.400 ± 4.030 84.480 ± 9.060 8 Prednisone 20.690 ± 2.640 7.600 ± 0.430 9 Progesterone 0.288 ± 0.026 0.409 ± 0.037 10 Testosterone 2.630 ± 0.280 5.670 ± 0.550 The peak and trough were observed at 390 and 420 nm, respectively, for various substrate concentrations. The peak-to-trough absorbance differences were plotted against the respective substrate concentrations for determining the
K d value using the equation Aobs = Amax (([S]+[Et]+KD) - (([S]+[Et]+KD)2 - (4[S][Et])0.5)/2[Et].
Effect of pH, Temperature, and Ionic Strength on Enzymatic Activity
The catalytic activity of an enzyme is highly dependent upon assay conditions such as pH, temperature, and ionic strength. The maximal activity of the purified CYP154C3s was observed in potassium phosphate buffer at pH 7.4. Both CYP154C3s showed good pH stability over the pH range of 7.0–7.8, retaining more than 92% of the maximal activity (Fig. 4A). The activity performance as a function of temperature was also very similar for both CYP154C3s. The optimal temperature range for both CYP154C3 activities was 30–37°C, retaining more than 98%of the maximal activity (Fig. 4B). The activity started to decrease dramatically at temperatures above 40°C. Since there is a strong electrostatic interaction between CYP and its redox partners, which is strongly based on charge pair interactions in addition to hydrophobic interactions [42, 43], the ionic strength dependency of the electron transfer from the redox partners to the CYPs was investigated. The catalytic activities of CYP154C3s in various ionic strength conditions maintained by NaCl (10–200 mM) were analyzed (Fig. 4C). The activity of both enzymes showed a marked bell-shaped dependence upon ionic strength. Lower ionic strength improved the enzymatic activity, reaching a maximum at 70 mM for both enzymes. Elevating the ionic strength above 70 mM decreased the enzymatic activity. Higher ionic strength disrupted the electrostatic interactions of the CYP redox partner complex, thereby decreasing the activity [44, 45].
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Fig. 4.
Optimum pH and temperature and the effect of ionic strength on CYP154C3-1 (■) and CYP154C3-2 (●). (A ) Optimum pH. The CYP154C3-1 and CYP154C3-2 activities were measured over a pH range of 6.0–8.5 at 30°C for 1 h. Both the CYPs favored alkaline pH (7.0–7.8), retaining more than 92% of the maximal activity. (B ) Optimum temperature. The activity of reactions at 15–50°C in pH 7.4 was measured for 1 h. At a 30–37°C temperature, the enzyme retained more than 98% of maximal activity. (C ) The effect of ionic strength on the catalytic activity of the enzymes in various ionic strength solutions maintained by NaCl (10–200 mM). A bell-shaped curve revealed improvement in activity in lower ionic strength, while higher ionic strength (>70 mM) decrease the enzyme activity. The hydroxylation of progesterone was measured. The values are the mean of three independent experiments with standard deviation.
Enzyme Kinetic Studies
To gain a deeper understanding of the enzymes, the overall kinetic parameters of purified CYP154C3s were evaluated by directly analyzing the product formation from a panel of substrates by HPLC. The measurements were carried out using purified CYP154C3s in the presence of the heterologous redox partners Pdx and PdR from
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Table 2 . Michaelis-Menten constant (
K m), maximum velocity (V max), the catalytic rate constant (k cat), and the coupling efficiency using purified CYP154C3s, PdR, Pdx, and 10 different steroid substrates.SN Substrate K m [μM]V max [μM Product / μM CYP / min]K cat [1/S]Coupling efficiency (%) CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 1 Adrenosterone 30.110 ± 1.711 27.850 ± 1.820 0.804 ± 0.013 0.793 ± 0.017 48.240 ± 0.780 47.580 ± 1.020 29.440 ± 3.770 31.800 ± 4.380 2 Androstenedione 38.480 ± 4.350 26.980 ± 1.540 0.844 ± 0.031 0.787 ± 0.014 50.640 ± 1.860 47.220 ± 0.840 27.430 ± 3.920 22.040 ± 3.830 3 Corticosterone 59.700 ± 6.350 81.170 ± 11.630 0.941 ± 0.031 0.923 ± 0.046 56.460 ± 2.100 55.380 ± 2.760 19.380 ± 3.280 19.880 ± 3.300 4 Cortisone 83.500 ± 6.670 108.510 ± 12.480 0.930 ± 0.027 0.979 ± 0.041 55.800 ± 1.620 58.740 ± 2.460 21.650 ± 3.390 23.410 ± 3.920 5 Hydrocortisone 141.420 ± 18.120 170.750 ± 22.780 0.231 ± 0.015 0.228 ± 0.015 13.860 ± 1.600 13.680 ± 1.900 13.320 ± 2.860 11.870 ± 3.060 6 Nandrolone 53.820 ± 7.170 49.830 ± 11.180 0.893 ± 0.043 0.821 ± 0.068 53.580 ± 2.280 49.260 ± 4.050 23.530 ± 3.470 24.990 ± 3.590 7 Prednisolone 246.540 ± 38.310 280.990 ± 37.510 0.294 ± 0.023 0.285 ± 0.020 17.640 ± 1.380 17.100 ± 1.200 8.770 ± 3.140 9.160 ± 2.720 8 Prednisone NA NA NA NA NA NA 25.130 ± 2.460 24.230 ± 3.750 9 Progesterone 6.320 ± 0.710 10.670 ± 1.220 0.833 ± 0.210 0.765 ± 0.029 49.980 ± 1.260 45.900 ± 1.740 39.490 ± 4.120 36.060 ± 4.280 10 Testosterone 10.380 ± 0.830 18.260 ± 3.220 0.933 ± 0.025 0.915 ± 0.052 55.980 ± 1.500 54.900 ± 3.120 35.440 ± 4.320 33.680 ± 4.170 The overall apparent kinetic parameters were determined with a CYP: Pdx: Pdr concentration ratio of 1: 8: 2 for the purified CYP154C3s toward 10 substrates. Coupling efficiency was calculated as the percentage of NADH used for the formation of product over the total NADH consumption. The NADH consumption rate was calculated after subtracting the respective background NADH consumption. The results represent the mean values of triplicate measurements.
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Fig. 5.
Hyperbolic fit for progesterone. The reaction was catalyzed by CYP154C3-1 (A ) and CYP154C3-2 (B ) using Pdx-Pdr-NADH. The reaction mixture contained CYP: Pdx: Pdr at a ratio of 1: 8: 2 with varied substrate concentration and the reaction was started by the addition of 250 μM NADH. The graph of rate of reaction vs substrate concentration was plotted. Data were analyzed by non-linear regression analysis based on Michaelis-Menten kinetics. The values are the mean of three independent experiments with standard deviation.
Coupling efficiencies are essential parameters for examining the efficiency of the catalytic system as a function of the used substrate. The coupling efficiency represents the percentage of NADH oxidized, which, in this case, was used for steroid hydroxylation. The coupling efficiencies of the substrates were calculated and the values were in the range of 8–39% and remained similar between the two CYP154C3s (Table 1). The coupling efficiency of
In Vitro Study of Steroid Hydroxylation
The activity and specificity of the recombinant CYP154C3s were evaluated by performing CYP-dependent substrate conversion assays using a panel of substrates and by subsequent HPLC and LC-MS analyses of the hydroxylated products (Table S1). The in vitro reactions catalyzed by CYP154C3s were conducted separately in three different reaction systems consisting of NADH, H2O2, and PIDA. The total conversion of the substrate by the catalytic activity of the enzymes using different reaction systems is shown in the heatmap in Fig. 6.
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Fig. 6.
Heat map of the total product formation using different redox systems (A, CYP: Pdx: Pdr at a ratio of 1: 8: 2; B, 3 mM PIDA; and C, 50 mM H2O2) with purified CYP154C3s. The reaction time was 2 h at 30°C. The percentage turnover was derived from the ratio of the product peak area to the sum of the substrate and product area on H PLC analysis. C3-1 and C3-2 represent CYP154C3-1 and CYP154C3-2, respectively. The number inside the box represents the percentage of products formed. The intensity of the color indicates the different substrate conversion ratios.
In the presence of NADH cofactor, the purified heterologous redox partners Pdx-PdR were able to transfer electrons to the CYP154C3s. The in vitro conversion of substrates showed hydroxylation in more than one position, displaying no stereo- or regioselectivity (Fig. S4). LC-MS analysis of the reaction mixtures of
The product analysis of a single monohydroxylated peak revealed possible hydroxylation at the C16 position. The presence of an ion mass of m/z 121 and 145 of the unmodified steroidal A/B ring indicates possible C16 hydroxylation [47, 48]. The single monohydroxylated peaks (P1) of
The conversion of the substrate by an alternate redox partner was also investigated by conducting an in vitro assay supported by the oxygen surrogates H2O2 and PIDA. The bacterial peroxygenases (CYP152 family) use H2O2 in the peroxide shunt pathway to catalyze reactions [8]. When using H2O2 in an in vitro assay, the oxidative degradation of heme by H2O2 is a major issue. Therefore, the H2O2 tolerance test was performed to determine the effect of H2O2 on the CYP154C3-mediated reaction. The test was performed using a range of H2O2 (0.2–200 mM) and observing the decrease in the Soret absorbance of the oxidized form of the CYP154C3s. Both CYP154C3s were active in high H2O2 concentrations (>10 mM). The optimal conversion of the substrates occurred at ~65 mM H2O2 and 3 mM PIDA (data not shown). Similar results for the optimal H2O2 and PIDA concentrations for CYP154C4 and CYP154C8 were reported [17, 25]. Additionally, it was also reported that the choice of suitable surrogate redox partners as well as reducing equivalents played an important role in the product distribution and catalytic efficiency of CYP enzymes [9].
The activity of CYP154C3s in the presence of H2O2 with steroid substrates was low with little change in the product distribution pattern (Fig. S4). For
The catalytic conversion of the substrates using PIDA was relatively low compared to Pdx/PdR but higher compared to H2O2 (Fig. S4). New products were observed with
CYP154C3s hydroxylated
CYP154C3 from
Conclusions
In summary, two CYPs from
Supplemental Materials
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1D1A3A03103903). In addition, this work was supported by the Korea Polar Research Institute (KOPRI grant number PM20030).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(3): 464-474
Published online March 28, 2021 https://doi.org/10.4014/jmb.2010.10020
Copyright © The Korean Society for Microbiology and Biotechnology.
Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 from Streptomyces Species
Pradeep Subedi1, Ki-Hwa Kim1, Young-Soo Hong2, Joo-Ho Lee3, and Tae-Jin Oh1,3,4*
1Department of Life Science and Biochemical Engineering, Sunmoon University, Asan 31460, Republic of Korea
2Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang-eup, Chungbuk 28116, Republic of Korea
3Genome-Based BioIT Convergence Institute, Asan 31460, Republic of Korea
4Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asan 31460, Republic of Korea
Correspondence to:Tae-Jin Oh,
tjoh3782@sunmoon.ac.kr
Abstract
Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3‐1) and Streptomyces sp. KCCM40643 (CYP154C3‐2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners’ putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C–C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.
Keywords: Steroid hydroxylase, Streptomyces, hydrogen peroxide, (diacetoxyiodo) benzene, C16 hydroxylation, C–,C bond cleavage
Introduction
The cytochrome P450s (CYPs) are a vast group of heme-containing enzymes found in virtually all living organisms. CYPs have a broad range of substrate specificity and are responsible for the hydroxylation of non-activated carbon atoms, dealkylation, epoxidation, demethylation, sulfoxidation, and carbon-carbon bond cleavage, making them an attractive target as biocatalysts for organic synthesis [1-5]. For catalysis, the majority of the microbial CYPs rely on one or more redox partners to supply redox equivalents. Cytochrome P450 reductases (CPRs) fused to a CYP domain as in the CYP102A and CYP116B family directly deliver electrons to the heme iron while NAD(P)H-dependent ferredoxin reductase (FDR) and ferredoxin (FDX) sequentially deliver electrons to the heme iron from the cofactor [6]. In addition, certain CYPs such as peroxygenases from the CYP152 family use hydrogen peroxide (H2O2) effectively, without the need for redox partners [7, 8]. Furthermore, some studies have used oxygen surrogates (OSs) to support CYP activity, although lower activity and rapid inactivation of the CYPs limited the efficiency of the OS-supported reactions [9, 10].
Steroids are ubiquitously available bioactive compounds with a wide range of therapeutic effects such as anti-inflammatory, immunosuppressive, anabolic, and diuretic activities [11-14]. Variations in the functional group, type, number, and attachment position to the core steroid subunit directly influence the biological function of steroids. Since steroids are hydrophobic, the presence of the hydroxyl group increases the polarity of the steroid. This may provide a higher level of biological activity or serve as an intermediate for the synthesis of steroidal derivatives. The presence of the hydroxyl group at the C7α position of dehydroepiandrosterone and pregnenolone displayed immunoprotective and immunoregulatory properties. Moreover, it was reported that the C11α-hydroxylation of deoxycortisone was crucial for anti-inflammatory activity, the 17β-hydroxyl function determined androgenic properties, and the 14β-hydroxyl group of steroids was cardio-active [15-19].
In eukaryotes and prokaryotes, steroid hydroxylation is accomplished exclusively by cytochrome P450 monooxygenases [20]. Bacterial monooxygenases compared to those in eukaryotes are advantageous for biotechnological applications because of their solubility and high level of expression [21]. A few CYPs are involved in the hydroxylation of steroids, like the CYP106A family (CYP106A1 and CYP106A2), the CYP109 family (CYP109B1 and CYP109E1), the CYP154C family (CYP154C2, CYP154C3, CYP154C4, CYP154C5, and CYP154C8), and CYP260A1 [17,22-27].
In this study, the cloning, heterologous overexpression, purification, and characterization of two CYP154C3s from
Materials and Methods
Materials
The steroids used in this study were obtained from TCI (Tokyo Chemical Industry Co., Ltd, Japan). Isopropyl-1-thio-β-D-galactopyranoside (IPTG), 1,4-dithiothreitol (DTT), and kanamycin were purchased from Duchefa Bohemie (Korea). Ampicillin, δ-aminolevulinic acid (ALA), NADH, and formate dehydrogenase were purchased from Sigma-Aldrich (Korea). All of the restriction enzymes, DNA polymerase, T4 DNA ligase, and dNTPs were purchased from Takara Bio (Japan). All other high-grade chemical products were obtained from commercially available sources.
Bioinformatics Analysis
The nucleotide sequences of CYP154C3-1 and CYP154C3‐2 were deposited in GenBank under accession numbers MF467273 and MT921810, respectively. Identification of the close homologs and comparison of the protein sequences were performed using the Basic Local Alignment Search Tool (BLAST). Multiple sequence alignment was accomplished using GeneDoc [28]. An evolutionary study was conducted using molecular evolutionary genetics analysis (MEGA X) [29]. A phylogenetic tree was constructed using the maximum likelihood method with 1000 bootstrap replicates and the evolutionary distances were computed using the Poisson correction method [30]. The CYP names were assigned by Dr. David Nelson (http://drnelson.utmem.edu/CytochromeP450.html).
Strains, Media, and Conditions
Molecular Cloning and Protein Over-Expression
The CYP154C3-1 and CYP154C3-2 encoding sequences (1,239 bp and 1,254 bp, respectively) were amplified from the genomic DNA of
For the in vitro reconstituted system, redox partners putidaredoxin (Pdx) and putidaredoxin reductase (PdR) were over-expressed and His-tagged in
Protein Purification and Concentration Determination
The crude extracts obtained by ultra-sonication were centrifuged at 12,500 rpm for 25 min at 4°C to remove cellular debris. The soluble fraction of the cell extracts was mixed with TALON His-tag resin pre-equilibrated with equilibrium buffer (potassium phosphate buffer pH 7.4) and shaken for 60 min. The protein-bound resin was eluted with elution buffers (potassium phosphate buffer pH 7.4 with 10% glycerol) containing 20 mM, 100 mM, and 250 mM imidazole. The fractions containing the proteins of interest were concentrated by ultrafiltration using Amicon centrifugal filters (Millipore) with a 30 kDa cutoff for CYP154C3s and PdR, whereas a 10 kDa cutoff was used for Pdx.
The concentration of the CYP154C3s was measured based on the CO-difference spectra method [32]. Using the potassium phosphate buffer protein, the protein was diluted to 2 ml and separated into two cuvettes (reference and sample). The sample cuvette was bubbled gently with carbon monoxide at a rate of 1 bubble per second for 1 min. Both the reference and sample were reduced with a few grains of sodium dithionite and the spectrum was recorded using a Biochrome Libra S35PC UV/Vis spectrophotometer (England). The concentration of functional CYP154C3s was estimated using an extinction coefficient ε450-490 of 91 mm-1 cm-1 [33]. The PdR concentration was estimated by calculating the average concentration from the 378 nm, 454 nm, and 480 nm wavelengths using extinction coefficients (ε) of 9.7, 10.0, and 8.5 mM-1cm-1, respectively [34]. The Pdx concentration was estimated by calculating the average concentration from the 415 nm and 454 nm wavelengths using extinction coefficients (ε) of 11.1 and 10.4 mM-1cm-1, respectively [31].
Effect of pH, Temperature, and Ionic Strength on Enzymatic Activity
The optimal pH for purified CYP154C3 activity was determined at 30°C using 50 mM potassium phosphate buffer with various pH ranges from 6.0 to 8.5. The maximal enzymatic activity at 30°C using phosphate buffer at pH 7.4 was defined as 100%. For the optimal temperature determination, the enzymatic activity assay was performed at various temperatures from 15–50°C in 50 mM phosphate buffer (pH 7.4). The maximum activity at 30°C in phosphate buffer (pH 7.4) was defined as 100%. The effect of the ionic strength maintained by sodium chloride (NaCl) on CYP154C3 activity was determined by adding different concentrations of NaCl (0–200 mM) into the reaction system. The maximal activity at 30°C in phosphate buffer (pH 7.4) with 70 mM NaCl was defined as 100%. Progesterone was used as the substrate for the characterization of both CYP154C3s in a reaction mixture containing 3 μM CYP154C3s, 100 μM substrate, 6 μM PdR, 24 μM Pdx, 100 μg/ml catalase, and 250 μM NADH. The reaction mixture was extracted twice with ethyl acetate and dried under vacuum for further analysis.
Enzymatic Activity Assay
The in vitro assay was carried out in the presence of NADH, H2O2, and PIDA. Ten substrates were used for the enzymatic activity assays (Fig. 1). The substrates were prepared by dissolving them in dimethyl sulfoxide (DMSO). The reaction mixture contained 3 μM CYP154C3s, 200 μM substrate, 6 μM PdR, 24 μM Pdx, 100 μg/ml catalase, and an NADH-regeneration system comprised of 1 U formate dehydrogenase, 150 mM sodium formate, and 1 mM MgCl2 in a final volume of 250 μl in 50 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of 250 μM NADH and incubated at 30°C for 2 h. The reaction mixture was extracted twice with 250 μl of ethyl acetate dried and analyzed by high-performance liquid chromatography (HPLC). Oxidizing agent (H2O2 and PIDA)-mediated conversion assays were also performed after determining the optimum concentration required for enzymatic activity. The reaction system consisted of 3 μM CYP154C3s and 200-μM substrate and was initiated by the addition of optimum concentrations of either H2O2 or PIDA. The reaction was incubated at 30°C for 2 h and extracted as described above.
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Figure 1. Structure of the steroids used to determine the substrate specificity of the CYP154C3s.
Determination of Kinetic Parameters and Catalytic Efficiency
A reaction curve was generated for both the CYP143C3s by measuring the product formed over time from the different substrates. Product quantification was achieved by correlating the peak area of the product(s) with the total peak area of the product(s) and the substrate. The reaction mixture typically contained 1 μM CYP154C3s, 2 μM PdR, 8 μM Pdx, and 100 μM substrate. The reactions were initiated by adding 350 μM NADH. After establishing the initial velocity conditions, the concentrations of all substrates were varied in the range of 0–500 μM to generate a saturation curve. The
NADH-Coupling Efficiency
NADH oxidation rates were measured spectrophotometrically using purified CYP, PdR, and Pdx by monitoring the NADH absorbance at 340 nm over time. The reaction mixture typically contained 1 μM CYPs, 2 μM PdR, 10 μM Pdx, and 200 μM substrate in 50 mM phosphate buffer at pH 7.4. The reactions were initiated by the addition of 250 μM NADH (ε = 6.22 mM-1 cm-1) [31]. The substrate consumption was determined by HPLC. The background NADH consumption in the absence of substrate was also determined. The coupling efficiency was calculated as the percentage of NADH used for product formation over the total NADH consumption.
Determination of the Substrate Dissociation Constant
Substrate binding assays were performed by the spectrophotometric titration of the enzymes (CYP154C3s) in 50 mM potassium phosphate buffer (pH 7.4) with increasing substrate concentrations until saturation. The absorbance spectra of all samples were recorded from 350 to 500 nm using a Biochrome Libra S35PC UV/Vis Spectrophotometer (England). The
In Eq. (1), Aobs is the absorption shift determined at any ligand concentration, Amax is the maximal absorption shift obtained at ligand saturation, [Et] is the enzyme concentration used, [S] is the substrate concentration, and KD is the apparent dissociation constant for the enzyme–ligand complex.
HPLC and LC-MS Analysis
After drying, the extracted reaction mixture was used for analysis. The dried residue was dissolved in HPLC-grade methanol, filtered through a 0.45‐μm pore polytetrafluoroethylene filter, and analyzed by ultra-high-performance liquid chromatography (UHPLC). The sample was injected and separated using a Mightysil reverse phase C18 column (4.6 × 250 mm, 5 μm). Acetonitrile (B) and water (A) were used as the mobile phase in a gradient system of B at 15% for 0–10 min, 50% for 10–20 min, 70% for 20–25 min, and 15% for 25–40 min at a flow rate of 1 ml/min. Detection of the substrates and their product was performed by UV absorbance at 242 and 245 nm. Liquid chromatography-mass spectroscopy (LC-MS) analysis was performed with a SYNAPT G2-S/ACUITY UPLC liquid chromatography quadrupole time-of-flight/electrospray ionization mass spectrometer (Waters, USA) in the positive ion mode. The products formed were identified by comparison to products reported previously [17, 25, 36].
Results and Discussion
Bioinformatics Analysis
Multiple sequence alignment of the selected proteins was performed to observe sequence conservation and the presence of the signature motif. The characteristic conserved oxygen-binding and activation I-helix motif, K helix (EXXR) motif, and heme-binding domain for the CYP family were observed (Fig. 2). All proteins contained an acid-alcohol pair, glutamate, and a threonine residue, which facilitates oxygen activation in CYPs [37]. Homologs of the protein sequences were searched by conducting a PSI-BLAST search (NCBI server). A phylogenetic tree was constructed using the protein sequences of the CYP154C3s and their closest homologs (Fig. 3). Phylogenetic analysis revealed that the CYP154C3s clustered closer to the previously studied
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Figure 2.
Multiple sequence alignment of The conserved and similar residues are highlighted. The highly conserved, functionally relevant regions (oxygen binding motif, EXXR motif, and heme‐binding signature motif) are underlined. The conserved cysteine residue of heme-binding motif is highlighted in red.Streptomyces CYP154Cs with CYP154C3, CYP154C4, and CYP154C5.
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Figure 3.
Phylogenetic tree of The phylogenetic tree was based on amino acid sequences of the CYP154 family. The phylogenetic tree was constructed by the maximum likelihood method and the Poisson correction model. The prefixes in front of the CYP number abbreviate the genus and the species of the respective bacteria from where the enzyme originated (Faln,Streptomyces CYP154C3s and their closest homologs.Frankia alni ; Nfar,Nocardia Farcinia ; Save,Streptomyces avermitilis ; Sbin,Streptomyces bingchengensis ; Scar,Streptomyces carzinostaticus ; Scoe,Streptomyces coelicolor ; Sfra,Streptomyces fradiae ; Sgri,Streptomyces griseus ; Speu,Streptomyces peucetius ; Sroc,Streptomyces rochei ; Ssca,Streptomyces scabies ; Sspp,Streptomyces sp.; Stro,Salinispora tropica ; and Tfus,Thermobifida fusca ). The P450s in the current study are indicated by the symbol (◈). The vertical bar in the tree represents 0.2 amino acid substitutions per amino acid for the branch length.
Cloning, Overexpression, Purification, and Spectral Characterization of Proteins
The DNA fragments encoding the CYP154C3s genes were PCR-amplified and cloned into the pET32a (+) expression vector. Both CYPs, CYP154C3‐1 and CYP154C3‐2, showed a better co-expression of target proteins in the soluble form in
The cytosolic purified fraction of the CYP154C3s showed spectral properties characteristic of CYP enzymes by UV–Vis absorption spectroscopy. The carbon monoxide-bound, dithionite-reduced form of the CYP154C3s exhibited absorption maxima at 449 nm, the characteristic signature of CYP heme in its Fe2+CO complex form [38].
Substrate-Binding Assay
The binding of the substrates to the active site of CYP was observed by the displacement of the heme water ligand (the sixth ligand to heme iron). This resulted in a shift of the ferric heme iron from a low-spin to a high-spin state (so-called type I shift) with a minimum Soret absorption of around 420 nm and a maximum of around 390 nm [39, 40]. All of the steroids were tested for a possible type I spin shift in the CYP154C3s. Upon binding to CYP154C3s, the steroids exhibited a type I shift with a maximum absorbance at 390 nm and a minimum at 420 nm (Fig. S1). By titrating different concentrations of the substrates until saturation and fitting to a nonlinear tight-binding quadratic equation, the dissociation constant (
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Table 1 . The dissociation constant (
K d) of the CYP154C3s for steroids..SN Substrate K d [μM]CYP154C3-1 CYP154C3-2 1 Adrenosterone 0.318 ± 0.026 0.443 ± 0.035 2 Androstenedione 0.351 ± 0.033 0.475 ± 0.054 3 Corticosterone 14.060 ± 2.570 44.490 ± 3.970 4 Cortisone 50.730 ± 6.140 26.880 ± 3.510 5 Hydrocortisone 80.390 ± 15.850 107.280 ± 13.860 6 Nandrolone 13.530 ± 2.620 6.580 ± 0.840 7 Prednisolone 45.400 ± 4.030 84.480 ± 9.060 8 Prednisone 20.690 ± 2.640 7.600 ± 0.430 9 Progesterone 0.288 ± 0.026 0.409 ± 0.037 10 Testosterone 2.630 ± 0.280 5.670 ± 0.550 The peak and trough were observed at 390 and 420 nm, respectively, for various substrate concentrations. The peak-to-trough absorbance differences were plotted against the respective substrate concentrations for determining the
K d value using the equation Aobs = Amax (([S]+[Et]+KD) - (([S]+[Et]+KD)2 - (4[S][Et])0.5)/2[Et]..
Effect of pH, Temperature, and Ionic Strength on Enzymatic Activity
The catalytic activity of an enzyme is highly dependent upon assay conditions such as pH, temperature, and ionic strength. The maximal activity of the purified CYP154C3s was observed in potassium phosphate buffer at pH 7.4. Both CYP154C3s showed good pH stability over the pH range of 7.0–7.8, retaining more than 92% of the maximal activity (Fig. 4A). The activity performance as a function of temperature was also very similar for both CYP154C3s. The optimal temperature range for both CYP154C3 activities was 30–37°C, retaining more than 98%of the maximal activity (Fig. 4B). The activity started to decrease dramatically at temperatures above 40°C. Since there is a strong electrostatic interaction between CYP and its redox partners, which is strongly based on charge pair interactions in addition to hydrophobic interactions [42, 43], the ionic strength dependency of the electron transfer from the redox partners to the CYPs was investigated. The catalytic activities of CYP154C3s in various ionic strength conditions maintained by NaCl (10–200 mM) were analyzed (Fig. 4C). The activity of both enzymes showed a marked bell-shaped dependence upon ionic strength. Lower ionic strength improved the enzymatic activity, reaching a maximum at 70 mM for both enzymes. Elevating the ionic strength above 70 mM decreased the enzymatic activity. Higher ionic strength disrupted the electrostatic interactions of the CYP redox partner complex, thereby decreasing the activity [44, 45].
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Figure 4.
Optimum pH and temperature and the effect of ionic strength on CYP154C3-1 (■) and CYP154C3-2 (●). (A ) Optimum pH. The CYP154C3-1 and CYP154C3-2 activities were measured over a pH range of 6.0–8.5 at 30°C for 1 h. Both the CYPs favored alkaline pH (7.0–7.8), retaining more than 92% of the maximal activity. (B ) Optimum temperature. The activity of reactions at 15–50°C in pH 7.4 was measured for 1 h. At a 30–37°C temperature, the enzyme retained more than 98% of maximal activity. (C ) The effect of ionic strength on the catalytic activity of the enzymes in various ionic strength solutions maintained by NaCl (10–200 mM). A bell-shaped curve revealed improvement in activity in lower ionic strength, while higher ionic strength (>70 mM) decrease the enzyme activity. The hydroxylation of progesterone was measured. The values are the mean of three independent experiments with standard deviation.
Enzyme Kinetic Studies
To gain a deeper understanding of the enzymes, the overall kinetic parameters of purified CYP154C3s were evaluated by directly analyzing the product formation from a panel of substrates by HPLC. The measurements were carried out using purified CYP154C3s in the presence of the heterologous redox partners Pdx and PdR from
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Table 2 . Michaelis-Menten constant (
K m), maximum velocity (V max), the catalytic rate constant (k cat), and the coupling efficiency using purified CYP154C3s, PdR, Pdx, and 10 different steroid substrates..SN Substrate K m [μM]V max [μM Product / μM CYP / min]K cat [1/S]Coupling efficiency (%) CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 1 Adrenosterone 30.110 ± 1.711 27.850 ± 1.820 0.804 ± 0.013 0.793 ± 0.017 48.240 ± 0.780 47.580 ± 1.020 29.440 ± 3.770 31.800 ± 4.380 2 Androstenedione 38.480 ± 4.350 26.980 ± 1.540 0.844 ± 0.031 0.787 ± 0.014 50.640 ± 1.860 47.220 ± 0.840 27.430 ± 3.920 22.040 ± 3.830 3 Corticosterone 59.700 ± 6.350 81.170 ± 11.630 0.941 ± 0.031 0.923 ± 0.046 56.460 ± 2.100 55.380 ± 2.760 19.380 ± 3.280 19.880 ± 3.300 4 Cortisone 83.500 ± 6.670 108.510 ± 12.480 0.930 ± 0.027 0.979 ± 0.041 55.800 ± 1.620 58.740 ± 2.460 21.650 ± 3.390 23.410 ± 3.920 5 Hydrocortisone 141.420 ± 18.120 170.750 ± 22.780 0.231 ± 0.015 0.228 ± 0.015 13.860 ± 1.600 13.680 ± 1.900 13.320 ± 2.860 11.870 ± 3.060 6 Nandrolone 53.820 ± 7.170 49.830 ± 11.180 0.893 ± 0.043 0.821 ± 0.068 53.580 ± 2.280 49.260 ± 4.050 23.530 ± 3.470 24.990 ± 3.590 7 Prednisolone 246.540 ± 38.310 280.990 ± 37.510 0.294 ± 0.023 0.285 ± 0.020 17.640 ± 1.380 17.100 ± 1.200 8.770 ± 3.140 9.160 ± 2.720 8 Prednisone NA NA NA NA NA NA 25.130 ± 2.460 24.230 ± 3.750 9 Progesterone 6.320 ± 0.710 10.670 ± 1.220 0.833 ± 0.210 0.765 ± 0.029 49.980 ± 1.260 45.900 ± 1.740 39.490 ± 4.120 36.060 ± 4.280 10 Testosterone 10.380 ± 0.830 18.260 ± 3.220 0.933 ± 0.025 0.915 ± 0.052 55.980 ± 1.500 54.900 ± 3.120 35.440 ± 4.320 33.680 ± 4.170 The overall apparent kinetic parameters were determined with a CYP: Pdx: Pdr concentration ratio of 1: 8: 2 for the purified CYP154C3s toward 10 substrates. Coupling efficiency was calculated as the percentage of NADH used for the formation of product over the total NADH consumption. The NADH consumption rate was calculated after subtracting the respective background NADH consumption. The results represent the mean values of triplicate measurements..
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Figure 5.
Hyperbolic fit for progesterone. The reaction was catalyzed by CYP154C3-1 (A ) and CYP154C3-2 (B ) using Pdx-Pdr-NADH. The reaction mixture contained CYP: Pdx: Pdr at a ratio of 1: 8: 2 with varied substrate concentration and the reaction was started by the addition of 250 μM NADH. The graph of rate of reaction vs substrate concentration was plotted. Data were analyzed by non-linear regression analysis based on Michaelis-Menten kinetics. The values are the mean of three independent experiments with standard deviation.
Coupling efficiencies are essential parameters for examining the efficiency of the catalytic system as a function of the used substrate. The coupling efficiency represents the percentage of NADH oxidized, which, in this case, was used for steroid hydroxylation. The coupling efficiencies of the substrates were calculated and the values were in the range of 8–39% and remained similar between the two CYP154C3s (Table 1). The coupling efficiency of
In Vitro Study of Steroid Hydroxylation
The activity and specificity of the recombinant CYP154C3s were evaluated by performing CYP-dependent substrate conversion assays using a panel of substrates and by subsequent HPLC and LC-MS analyses of the hydroxylated products (Table S1). The in vitro reactions catalyzed by CYP154C3s were conducted separately in three different reaction systems consisting of NADH, H2O2, and PIDA. The total conversion of the substrate by the catalytic activity of the enzymes using different reaction systems is shown in the heatmap in Fig. 6.
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Figure 6.
Heat map of the total product formation using different redox systems (A, CYP: Pdx: Pdr at a ratio of 1: 8: 2; B, 3 mM PIDA; and C, 50 mM H2O2) with purified CYP154C3s. The reaction time was 2 h at 30°C. The percentage turnover was derived from the ratio of the product peak area to the sum of the substrate and product area on H PLC analysis. C3-1 and C3-2 represent CYP154C3-1 and CYP154C3-2, respectively. The number inside the box represents the percentage of products formed. The intensity of the color indicates the different substrate conversion ratios.
In the presence of NADH cofactor, the purified heterologous redox partners Pdx-PdR were able to transfer electrons to the CYP154C3s. The in vitro conversion of substrates showed hydroxylation in more than one position, displaying no stereo- or regioselectivity (Fig. S4). LC-MS analysis of the reaction mixtures of
The product analysis of a single monohydroxylated peak revealed possible hydroxylation at the C16 position. The presence of an ion mass of m/z 121 and 145 of the unmodified steroidal A/B ring indicates possible C16 hydroxylation [47, 48]. The single monohydroxylated peaks (P1) of
The conversion of the substrate by an alternate redox partner was also investigated by conducting an in vitro assay supported by the oxygen surrogates H2O2 and PIDA. The bacterial peroxygenases (CYP152 family) use H2O2 in the peroxide shunt pathway to catalyze reactions [8]. When using H2O2 in an in vitro assay, the oxidative degradation of heme by H2O2 is a major issue. Therefore, the H2O2 tolerance test was performed to determine the effect of H2O2 on the CYP154C3-mediated reaction. The test was performed using a range of H2O2 (0.2–200 mM) and observing the decrease in the Soret absorbance of the oxidized form of the CYP154C3s. Both CYP154C3s were active in high H2O2 concentrations (>10 mM). The optimal conversion of the substrates occurred at ~65 mM H2O2 and 3 mM PIDA (data not shown). Similar results for the optimal H2O2 and PIDA concentrations for CYP154C4 and CYP154C8 were reported [17, 25]. Additionally, it was also reported that the choice of suitable surrogate redox partners as well as reducing equivalents played an important role in the product distribution and catalytic efficiency of CYP enzymes [9].
The activity of CYP154C3s in the presence of H2O2 with steroid substrates was low with little change in the product distribution pattern (Fig. S4). For
The catalytic conversion of the substrates using PIDA was relatively low compared to Pdx/PdR but higher compared to H2O2 (Fig. S4). New products were observed with
CYP154C3s hydroxylated
CYP154C3 from
Conclusions
In summary, two CYPs from
Supplemental Materials
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1D1A3A03103903). In addition, this work was supported by the Korea Polar Research Institute (KOPRI grant number PM20030).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

Fig 6.

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Table 1 . The dissociation constant (
K d) of the CYP154C3s for steroids..SN Substrate K d [μM]CYP154C3-1 CYP154C3-2 1 Adrenosterone 0.318 ± 0.026 0.443 ± 0.035 2 Androstenedione 0.351 ± 0.033 0.475 ± 0.054 3 Corticosterone 14.060 ± 2.570 44.490 ± 3.970 4 Cortisone 50.730 ± 6.140 26.880 ± 3.510 5 Hydrocortisone 80.390 ± 15.850 107.280 ± 13.860 6 Nandrolone 13.530 ± 2.620 6.580 ± 0.840 7 Prednisolone 45.400 ± 4.030 84.480 ± 9.060 8 Prednisone 20.690 ± 2.640 7.600 ± 0.430 9 Progesterone 0.288 ± 0.026 0.409 ± 0.037 10 Testosterone 2.630 ± 0.280 5.670 ± 0.550 The peak and trough were observed at 390 and 420 nm, respectively, for various substrate concentrations. The peak-to-trough absorbance differences were plotted against the respective substrate concentrations for determining the
K d value using the equation Aobs = Amax (([S]+[Et]+KD) - (([S]+[Et]+KD)2 - (4[S][Et])0.5)/2[Et]..
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Table 2 . Michaelis-Menten constant (
K m), maximum velocity (V max), the catalytic rate constant (k cat), and the coupling efficiency using purified CYP154C3s, PdR, Pdx, and 10 different steroid substrates..SN Substrate K m [μM]V max [μM Product / μM CYP / min]K cat [1/S]Coupling efficiency (%) CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 CYP154C3-1 CYP154C3-2 1 Adrenosterone 30.110 ± 1.711 27.850 ± 1.820 0.804 ± 0.013 0.793 ± 0.017 48.240 ± 0.780 47.580 ± 1.020 29.440 ± 3.770 31.800 ± 4.380 2 Androstenedione 38.480 ± 4.350 26.980 ± 1.540 0.844 ± 0.031 0.787 ± 0.014 50.640 ± 1.860 47.220 ± 0.840 27.430 ± 3.920 22.040 ± 3.830 3 Corticosterone 59.700 ± 6.350 81.170 ± 11.630 0.941 ± 0.031 0.923 ± 0.046 56.460 ± 2.100 55.380 ± 2.760 19.380 ± 3.280 19.880 ± 3.300 4 Cortisone 83.500 ± 6.670 108.510 ± 12.480 0.930 ± 0.027 0.979 ± 0.041 55.800 ± 1.620 58.740 ± 2.460 21.650 ± 3.390 23.410 ± 3.920 5 Hydrocortisone 141.420 ± 18.120 170.750 ± 22.780 0.231 ± 0.015 0.228 ± 0.015 13.860 ± 1.600 13.680 ± 1.900 13.320 ± 2.860 11.870 ± 3.060 6 Nandrolone 53.820 ± 7.170 49.830 ± 11.180 0.893 ± 0.043 0.821 ± 0.068 53.580 ± 2.280 49.260 ± 4.050 23.530 ± 3.470 24.990 ± 3.590 7 Prednisolone 246.540 ± 38.310 280.990 ± 37.510 0.294 ± 0.023 0.285 ± 0.020 17.640 ± 1.380 17.100 ± 1.200 8.770 ± 3.140 9.160 ± 2.720 8 Prednisone NA NA NA NA NA NA 25.130 ± 2.460 24.230 ± 3.750 9 Progesterone 6.320 ± 0.710 10.670 ± 1.220 0.833 ± 0.210 0.765 ± 0.029 49.980 ± 1.260 45.900 ± 1.740 39.490 ± 4.120 36.060 ± 4.280 10 Testosterone 10.380 ± 0.830 18.260 ± 3.220 0.933 ± 0.025 0.915 ± 0.052 55.980 ± 1.500 54.900 ± 3.120 35.440 ± 4.320 33.680 ± 4.170 The overall apparent kinetic parameters were determined with a CYP: Pdx: Pdr concentration ratio of 1: 8: 2 for the purified CYP154C3s toward 10 substrates. Coupling efficiency was calculated as the percentage of NADH used for the formation of product over the total NADH consumption. The NADH consumption rate was calculated after subtracting the respective background NADH consumption. The results represent the mean values of triplicate measurements..
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