Article Figures & Data
Figures
- Fig. 1.
Comparison of the hypothetical mRNA secondary structures, ΔG values, and P450 spectral characteristics expression of native CYP2S1dh, semiCYP2S1dh, and syn2S1dh. A, structures and ΔG values were calculated at a 37°C folding temperature and 1 M Na+ using mfold (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi). Expression of the native CYP2S1 construct (B), semiCYP2S1-dh construct (C), and syn2S1-dh construct (D) in E. coli LMG 194. All constructs were coexpressed with P450-reductase. Fe2+ CO versus Fe2+ difference spectra were measured using 1:5 dilutions of lysate supernatants. The expression levels of CYP2S1, semiCYP2S1-dh, and syn2S1-dh were estimated to be 20 to 30, 50 to 60, and 200 nM culture, respectively.
- Fig. 2.
Amino acid sequence alignment of human CYP2S1 with other CYP2 family enzymes. The highlighted amino acids are thought to be important in the interaction between the cytochromes P450 and P450 reductase.
- Fig. 3.
NADPH reduced Fe2+ CO versus Fe2+ difference spectra of CYP2S1 + P450 reductase (A). Light blue, CYP2S1 + CO; brown, CYP2S1 + CO + NADPH; blue, CYP2S1 + CO + NADPH + naphthalene; purple, CYP2S1 + CO + NADPH + naphthalene + dithionite. Spectra of CYP1A1 + P450 reductase (B): green, CYP1A1 + CO; blue, CYP1A1 + CO + NADPH; red, CYP1A1 + CO + NADPH + harmaline. Spectra of CYP2C9 + P450 reductase (C): black, CYP2C9 + CO; blue, CYP2C9 + CO + NADPH; light blue, CYP2C9 + CO + NADPH + diclofenac; green, CYP2C9 + CO + dithionite. Spectra of CYP3A4 + P450 reductase (D), green, CYP3A4 + CO; red, CYP3A4 + CO + NADPH; purple, CYP3A4 + CO + NADPH + diclofenac; blue, CYP3A4 + CO + NADPH + testosterone.
- Fig. 4.
Cumene hydroperoxide and hydrogen peroxide support oxidation by CYP2S1. HPLC chromatograms of 0.15 μM CYP3A4 + reductase (A) incubated with 100 μM testosterone and 1 mM cumene hydroperoxide (dark line), or with 200 μM testosterone and a NADP/NADPH regeneration mixture (gray line) for 10 min at 37°C. The 19-min peak corresponds to a 6-β-OH-testosterone, detected at 245 nm. HPLC chromatograms (B) comparing activities of 0.15 μM CYP2S1 (solid black line), 0.15 μM CYP1A1 (gray line), and human reductase sample (HR, dotted line) with 100 μM benzo[a]pyrene in the presence of 1 mM cumene hydroperoxide for 10 min at 37°C. HPLC chromatograms (C) of 0.15 μM CYP2S1, 0.15 μM CYP1A1, and human reductase sample (HR) toward 100 μM benzo[a]pyrene in the presence of 10 mM H2O2 for 10 min at 37°C. Products, indicated by arrows, were detected at 254 nm.
- Fig. 5.
Identification of CYP2S1-catalyzed products of BaP. HPLC chromatogram of 0.2 μM CYP2S1 incubated with 200 μM BaP and 1 mM CHP (solid black line). Products were identified at 254 nm using authentic standards (dotted lines), 6,12-BaP-quinone (4); 3,6-BaP-quinone (3); 1,6 benzo[a]pyrene (2), 4-hydroxy benzo[a]pyrene (4-OH-BaP) (5), and BaP-7,8-diol (1). Other products, indicated with question mark, were not identified.
- Fig. 6.
Identification of CYP2S1-catalyzed products of BaP-7,8-diol. HPLC chromatograms of 0.2 μM CYP2S1 incubated with 133 μM BaP-7,8-diol and 1 mM CHP (A), no CHP control (B), and no CYP2S1control (C). Detection was set at 345 nm. r7,t8,t9,c10-tetrol was identified using an authentic standard (D). Other unknown products, indicated by arrows, were also detected. The product marked with * has the same UV spectrum as r7,t8,t9,c10-tetrol, suggesting that it is its enantiomer.
- Fig. 7.
Identification of CYP2S1-catalyzed products of 9,10-H2-BaP. HPLC chromatogram of 0.2 μM CYP2S1 incubated with 133 μM 9,10-H2-BaP and 1 mM CHP (A), without CYP2S1 (C) control, or without CHP (B) control. Detection was set at 310 nm. The 7,8-diol-9,10-H2-BaP product was identified based on the comparison of its UV-spectrum (D) to that of r7,t8,t9,c10-tetrol (E).
- Fig. 8.
Identification of CYP2S1-catalyzed products of DMBA. HPLC chromatogram of 0.2 μM DMBA incubated with 500 μM DMBA and CHP (solid black). Products were identified at 295 nm using authentic standards, 12-OH-DMBA (3), 7-OH-DMBA (2), and 3,4-diol DMBA (1). Other unknown products are indicated by question marks.
- Fig. 9.
Identification of CYP2S1-catalyzed products of all-trans-retinoic acid (AtR). HPLC chromatograms of 0.2 μM CYP2S1 incubated with 20 μM AtR and 1 mM CHP (A) or 1 mM H2O2 (B), and of 0.1 μM CYP2C8 + reductase incubated with 5 μM AtR and NADPH (C). Products were detected at 350 nm. 4-OH-AtR was identified based on a previous report in which CYP2C8 produced 4-OH-AtR from AtR (Nadin and Murray, 1999).
- Fig. 10.
Identification of CYP2S1-catalyzed products of aflatoxin B1. HPLC chromatograms of 0.2 μM CYP2S1 incubated with 243 μM aflatoxin B1 and 3 mM H2O2 (i), of 0.2 μM CYP3A4 + reductase incubated with 243 μM aflatoxin B1 and NADPH (ii), and of CYP1A2 + reductase incubated with 243 μM aflatoxin B1 and NADPH (iii). Products were detected with UV-Vis detector at 360 nm (A) and with a fluorescence detector (B). M1 and Q1 were identified based on a previous report (Gallagher et al., 1996). Unknown products are indicated by arrows.
Tables
- TABLE 1
Km values and turnover numbers of various substrates using purified synthetically encoded CYP2S1 (0.2 μM) and CHP (1 mM)
Data are represented as averages ± S.E. of duplicate determinations. Km values were determined with Prism software using nonlinear regression with one site binding mode and the V = Vmax · S/(Km + S) equation. Turnovers were determined using the highest concentration of substrate.
Substrate Product Km Turnover μM nmol product/nmol CYP2S1/min Benzo[a]pyrene 1,6-Dione 94 ± 22 0.83 3,6-Dione 48.6 ± 8 1.85 6,12-Dione 72.7 ± 14 3.25 7,12-Dimethylbenz[a]anthracene 7-OH-DMBA 219 ± 4 15.7 12-OH-DMBA 34 ± 9 26 9,10-Dihydro-benzo[a]pyrene 9,10H2-BaP-7,8-dihydrodiol 22.6 ± 6 1.6 Benzo[a]pyrene-trans-7,8dihydrodiol r7,t8,t9,c10-tertol 12.43 ± 2 2.3 Aflatoxin B1 Unknown 266 ± 40 N.D. all-trans-Retinoic acid 4-OH-retinoic acid 20.9 ± 0.9 N.D. Naphthalene Unknown N.D. N.D. Styrene Unknown N.D. N.D. Testosterone No activity Coumarin No activity -
N.D., not determined.
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- TABLE 2
Km values and turnover numbers of CHP and H2O2 for various substrates, determined by incubation of 0.2 μM CYP2S1 and fixed concentration of substrates
Data are represented as averages ± S.E. of duplicate determinations.
Substrate Product CHP H202 Km Turnover Km Turnover μM nmol product/nmol CYP2S1/min μM nmol product/nmol CYP2S1/min Benzo[a]pyrene (167 μM) 1,6-Dione 16.1 ± 3.2 0.58 107 ± 21 0.44 3,6-Dione 29.4 ± 5 1.36 215 ± 42 0.8 6,12-dione 30.8 ± 6 2.24 232 ± 46 1.46 7,12-Dimethylbenz[a]anthracene (133 μM) 7-OH-DMBA 58.6 ± 11 15.7 4800 ± 950 15 12-OH-DMBA 62.0 ± 12 26 > 5000 14.8 9,10-Dihydro-benzo[a]pyrene (50 μM) 9,10H2-BaP-7,8-dihydrodiol 188 ± 37 0.55 60 ± 12 0.13 Benzo[a]pyrenetrans7,8dihydrodiol (133 μM) r7,t8,t9,c10-tertol 31.6 ± 5 1.1 1906 ± 350 0.3 Aflatoxin B1 (243 μM) Unknown 8.8 ± 2 N.D. 3500 ± 700 N.D. Retinoic acid (20 μM) 1.1 ± 0.4 N.D. 1.2 ± 0.4 N.D. -
N.D., not determined.
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Data Supplement
Files in this Data Supplement:
- Data Supplement -
Supplemental Material
Table S1. N-terminal modifications.
Table S2. Primers for N-terminal modifications of CYP2S1.Figure S1. Amino acid and DNA sequences of synthetic CYP2S1 (syn2S1).
Figure S2. Vector Map of pBdtacHR-h2S1 construct.
Figure S3. Expression of CYP2S1 modification 1 (A), 2 (B), 3 (C), and 4 (D) in E. coli.
Figure S4. Modifications introduced into the CYP2S1 cDNA to optimize codon usage in E. coli and minimize mRNA secondary structure.
Figure S5. Purification of synthetic CYP2S1 using a Ni2+ nitriloacetic acid (NTA) column with and without detergent.
- Data Supplement -
Supplemental Material
