1
Supporting Information
2
Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium
3
smegmatis
4
5
Hye-Yeong Jo, Sun-Ha Park, Thien-Kim Le, Sang Hoon Ma, Donghak Kim, Taeho Ahn, Young Hee
6
Joung, and Chul-Ho Yun*
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8
*Corresponding author: Professor Chul-Ho Yun, School of Biological Sciences and Technology,
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Chonnam National University, 77 Yongbongro, Gwangju 61186, Republic of Korea. Tel: +82-62-530-
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2194; Fax: +82-62-530-2199; E-mail: [email protected]
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Supplementary Methods
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Preparation of reductase systems
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Recombinant rat NADPH-P450 reductase (rCPR) (Hanna et al. 1998), Candida CPR (CaCPR) (Park
14
et al. 2010), and putidaredoxin reductase/putidaredoxin (Pdx/PDR) (Kim and Ortiz de Montellano
15
2009) were expressed in E. coli and purified as previously reported.
16
Construction of an expression plasmid for the CYP191A1 gene
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The M. smegmatis strain mc2155 was obtained from the Korean Collection for Type Culture (KCTC)
18
(Daejeon, Korea). After identifying the DNA sequence of the CYP191A1 enzyme from the M.
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smegmatis complete genome sequence (Accession number: CP000480.1) through the National Center
20
for Biotechnology Information (NCBI), the cDNA sequence was modified to include an NdeI
21
restriction
22
gataagctttcaatgatggagatgatggagcacctcgaccggcacgt) at the 5’- and 3’-ends, respectively. PCRs were
23
performed using pfu DNA polymerase and an MJ Research PTC-200 Thermal Cycler (Reno, NV).
24
The 1251-bp PCR product was resolved on a 1% (v/v) agarose gel, purified, digested with NdeI and
25
HindIII, and ligated into a pCWOri plasmid (Yun et al. 2006) that had been digested with the same
26
endonucleases. The recombinant pCW vector containing CYP191A1 was expressed in E. coli. Cloned
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CYP191A1 has been verified by full sequencing to eliminate any possible mutations (Intron
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Biotechnology, Gyeonggi-do, Korea).
29
Heterologous expression and purification of recombinant CYP191A1
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The plasmids were transformed into E. coli DH5α F’-IQ cells. Prior to starting the expression culture,
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the transformed cells were grown overnight in lysogeny broth (LB) with ampicillin (100 μg/ml) at
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37°C. The overnight cultures (2 ml) were used to inoculate a 200 ml culture of Terrific broth (TB)
33
containing 100 μg/ml ampicillin, 1 mM thiamine, trace elements, 50 μM FeCl3, 1 mM MgCl2, and 2.5
site
(5’-ggtcatatgaccacgacgg)
and
2
a
HindIII
(His)6
site
(5’
-
34
mM (NH4)2SO4. The cultures were grown at 37°C and 200 rpm to an OD600 between 0.4 and 0.6.
35
Following the addition of IPTG (1 mM) and δ-aminolevulinic acid (δ-ALA) (0.5 mM), the cultures
36
were grown at 32°C and 200 rpm for 32 h. The cells were harvested by centrifugation (15 min,
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5,000×g, 4°C). The cell pellet was resuspended in TES buffer [100 mM Tris/HCl (pH 7.6), 500 mM
38
sucrose, 0.5 mM EDTA] and lysed by sonication (Sonicator, Heat Systems – Ultrasonic, Inc.). After
39
the lysate was centrifuged at 100,000×g (90 min, 4°C), the soluble cytosolic fraction was collected
40
and dialyzed against 50 mM Tris/HCl (pH 7.4) and 0.1 mM EDTA. The cytosolic fraction was then
41
loaded onto a 2.5 × 10 cm Ni2+ Sepharose High Performance column that had been pre-equilibrated
42
with 50 mM Tris/HCl buffer (pH 7.4) containing 0.5 M NaCl and 5 mM imidazole. Contaminating
43
proteins were removed by extensive washing with equilibration buffer containing 20 mM imidazole.
44
Recombinant CYP191A1 enzymes were eluted from the column with 50 mM Tris/HCl buffer (pH
45
7.4) containing 300 mM imidazole and 0.5 M NaCl. Fractions containing P450 were pooled and
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dialyzed at 4°C for 4 h against a 200-fold volume of 50 mM Tris/HCl buffer (pH 7.4) containing 0.1
47
mM EDTA, followed by two more changes of the same buffer. SDS–PAGE was used to assess final
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protein purity, and P450 concentrations were determined by Fe2+–CO versus Fe2+ difference
49
spectroscopy (Omura and Sato, 1964). The final specific content of the purified CYP191A1 was 12
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nmol P450/mg protein.
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CYP191A1 catalytic activity assays
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Hydroxylation assay of fatty acids
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The fatty acid hydroxylation assay was performed as previously described (Jang et al. 2016;
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Gustafsson et al. 2004). 2 mM lauric acid, capric acid, myristic acid, or palmitic acid was included in
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the reaction mixture. The fatty acid stock solutions (200 mM) were prepared using dimethyl sulfoxide
56
(DMSO) and diluted into the enzyme reactions to a final organic solvent concentration of <1% (v/v).
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The reactions were performed for 5 min at 37°C and stopped with 50 μl 20% (w/v) trichloroacetic
3
58
acid (TCA). These steps were followed by the addition of 10-hydrodecanoic acid as an internal
59
standard for derivatization and extraction with a 2-fold excess of ice-cold dichloromethane (CH2Cl2).
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After centrifugation of the reaction mixture at 4°C (3,000 g, 5 min), the organic layer was transferred
61
to a clean glass tube, and the CH2Cl2 was removed under an N2 stream. The metabolites of the fatty
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acids were dissolved in BSTFA (70 μl) containing trimethylchlorosilane (TMCS) (1%, v/v). The
63
solution was transferred to a glass vial and then incubated at 75°C for 20 min to yield the
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trimethylsilylated products. To determine the regioselectivity at the ω-1, ω-2 and ω-3 positions of the
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hydroxylated products of the fatty acid metabolism, a GC analysis was performed using a Shimadzu
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QP2010 instrument (column length, 30 m; internal diameter, 0.25 mm; film thickness, 0.1 μm) with
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electron-impact ionization. The oven was maintained at 70°C for 1 min and then increased to 170°C
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at 25°C/min, to 200°C at 5°C/min, and to 280°C at 20°C/min. The oven was finally maintained at
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280°C for 5 min. The turnover numbers of the fatty acid hydroxylation was determined using a GC-
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FID detector (Shimadzu GC2010 with FID detector). The distribution of the products was based on
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the relative peak area of the GC chromatogram using hydroxylated products as standards.
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Hydroxylation assay of the chromogenic substrates
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4-Nitrophenol (4-NP) (Chang et al. 2006) hydroxylation activity of CYP191A1 was determined as
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described previously with slight modifications. Briefly, 800 μM 4-NP was included in the reaction
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mixtures of 0.5 ml. The stock solutions of both substrates (80 mM 4-NP) were prepared in DMSO.
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The reactions were performed for 5 min at 37°C, and the reaction was terminated by the addition of
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0.1 ml of 20% TCA. After centrifugation (~3000 g, 5 min), 200 μl of the organic layer from each
78
incubation were transferred to a 96-well plate with 0.1 ml 2 M NaOH. The absorbance was then
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measured at 510 nm for 4-NP using a microtiterplate reader.
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Oxidation of coumarin and 7-EC
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The oxidation assays of coumarin and 7-EC were performed as previously described (Yun et al. 2005;
4
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Kim et al. 2008). Coumarin or 7-EC was added to the reaction mixtures at 1 mM. The stock solutions
83
of both substrates (100 mM) were prepared in CH3CN. The reactions were performed for 5 min at
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37°C, and the reaction was terminated by the addition of 0.5 ml of ice-cold CH2Cl2. After
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centrifugation (3,000 g, 5 min), 300 μl aliquots of the organic layer from each incubation were
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transferred to a clean glass tube, and the CH2Cl2 was removed under a gentle stream of N2 gas. The
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metabolites of 7-EC and coumarin were analyzed via HPLC using a Gemini C18 column (4.6 × 150
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mm, 5 μm; Phenomenex, Torrance, CA, USA) with a mobile phase of H2O/CH3CN (55:45, v/v)
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containing 10 mM HClO4. The flow rate was 1 ml/min, and the absorbance was monitored at A254.
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7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation assay
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The 7-EFC O-deethylation activity was measured using a previously described fluorescence assay
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(Kim et al. 2002; Buters et al. 1993). Briefly, 50 μM 7-EFC was added to the reaction mixtures. A
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stock solution of the substrate (5 mM) was prepared in DMSO. The reaction mixtures were incubated
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for 5 min at 37°C, and the reactions were terminated by the addition of 25 μl 20% TCA. After the
95
addition of 400 μl of 0.1 M Tris/HCl (pH 9), the absorbance of the mixture was measured using a
96
spectrofluorometer. HFC was used as a standard (excitation at 410 nm and emission at 510 nm).
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Alkoxyresorufin O-dealkylation
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The activities of 7-ethoxyresorufin O-deethylation (EROD) were measured using a fluorescence assay
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(Burke et al. 1985). The reaction mixture contained 5 μM 7-ethoxyresorufin. This mixture was
100
incubated for 5 min at 37°C, and the reaction was terminated by the addition of 500 μl of methanol.
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The metabolites were analyzed using fluorescence and a resorufin standard (excitation at 535 nm and
102
emission at 585 nm).
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The O-deethylation activities of 7-methoxyresorufin and 7-pentoxyresorufin were also
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measured using a fluorescence assay in the presence of peroxide. The reaction mixture contained 5
105
μM MR or PR. The reaction was performed for 5 min at 37°C and terminated by the addition of 1 ml
5
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of methanol. After centrifugation (~3000 g, 10 min), the metabolites were analyzed using
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fluorescence and a resorufin standard (excitation at 535 nm and emission at 585 nm).
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Hydroxylation of drug substrates supported by peroxide
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The oxidation of simvastatin, lovastatin, and atorvastatin was analyzed as previously described (Kim
110
et al. 2011; Kang et al. 2014). The statins were added to the reaction mixtures at a concentration of 80
111
μM. A stock solution of the statins (200 mM) was prepared in DMSO. The reaction mixtures were
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incubated for 5 min at 37°C, and the reactions were terminated by the addition of a 2-fold excess of
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ice-cold CH2Cl2. After centrifugation of the reaction mixture (3000 rpm, 5 min), the organic layer was
114
transferred to a clean glass tube, and the CH2Cl2 was removed under a N2 stream. The metabolites
115
were analyzed via HPLC using a Gemini C18 column (4.6 × 250 mm, 5 μm; Phenomenex, Torrance,
116
CA, USA) with an acetonitrile/water (70:30, v/v) mobile phase containing 2.5 mM formic acid. The
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eluate was monitored using UV spectroscopy at 240 and 275 nm (using dual mode).
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The oxidation of resveratrol was measured as previously described (Kim et al. 2009). The
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reaction mixture contained 100 μM resveratrol. The reaction mixtures were incubated for 5 min at
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37°C, and the reactions were terminated by the addition of a 2-fold excess of ethyl acetate. After
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centrifugation (3000 rpm, 10 min), the organic layer was transferred to a clean glass tube, and the
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ethyl acetate was removed under a N2 stream. The reaction products were analyzed via HPLC using a
123
Gemini C18 column (4.6 × 150 mm, 5 μm; Phenomenex, Torrance, CA, USA). The mobile phase
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consisted of water and 0.5% acetic acid/acetonitrile (95:5 v/v) for buffer A and acetonitrile/water and
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0.5% acetic acid (95:5, v/v) for buffer B. The flow rate was 1 ml/min (isocratic flow), and the
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absorbance of the eluate was monitored at A320.
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The chlorzoxazone hydroxylation assay was performed as described preciously (Guengerich
128
et al. 1991). A stock solution of chlorzoxazone (20 mM) was prepared in 60 mM KOH. The reaction
129
mixture contained 200 μM chlorzoxazone. The reaction mixtures were incubated for 5 min at 37°C,
6
130
and the reaction was stopped by the addition of a 2-fold excess of 43% H3PO4 and extracted with a 2-
131
fold excess of ice-cold CH2Cl2. After centrifugation of the reaction mixture (3000 rpm, 5 min), the
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organic layer was transferred to a clean glass tube, and the CH2Cl2 was removed under an N2 stream.
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The metabolites were analyzed via HPLC using a Luna C8 column (4.6 × 150 mm, 5 μm;
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Phenomenex, Torrance, CA, USA) with a mobile phase of H2O/CH3CN/H3PO4 (75.5:27:0.5,v/v/v)
135
and a flow rate of 1.2 ml/min. The absorbance of the eluate was monitored at A287.
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The phenacetin oxidation assay was performed as preciously described (Yun et al. 2000). The
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reaction mixture contained 1 mM phenacetin. A stock solution of phenacetin (100 mM) was prepared
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in 60 mM KOH. The reaction mixtures were incubated for 5 min at 37°C, and the reactions were
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terminated by the addition of a 2-fold excess of ice-cold CH2Cl2 followed by the addition of 0.5 ml of
140
a mixture of CHCl3:2-propanol (6:4, v/v). After centrifugation of the reaction mixture (3000 rpm, 10
141
min), the organic layer (CHCl3:2-propanol (6:4, v/v)) was transferred to a clean glass tube, and the
142
solvent was removed under an N2 stream. The metabolites were analyzed via HPLC using a Gemini
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C18 column (4.6 × 250 mm, 5 μm; Phenomenex, Torrance, CA, USA) with a H 2O/CH3CN/H3PO4
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(65:35:0.1, v/v/v) mobile phase and a flow rate of 0.8 ml/min. The absorbance of the eluate was
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monitored at A254.
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Spectroscopy
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The UV-visible spectra were recorded using a Shimadzu UV-1601 instrument (Shimadzu, Kyoto,
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Japan) at room temperature. The spectra of the ferric, sodium dithionite–reduced ferrous, and ferrous–
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CO complexes of CYP191A1 were recorded in 100 mM potassium phosphate buffer (pH 7.4). The
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high-spin contents of the CYP191A1 enzymes were estimated from the second-derivative spectra of
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the ferric enzymes, as described previously (Guengerich, 1983). In all of the fluorescence experiments,
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the emission spectra were recorded using a Shimadzu RF-5301 PC spectrofluorometer equipped with
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a thermostated cuvette compartment.
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7
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Supplementary Table 1. Comparison of the activities of CYP191A1-catalyzed oxidations to the
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marker activities of human P450 isoforms.
Substrate
Reaction
CYP191A1
(min-1) with
a best redox
system
7-Ethoxycoumarin
O-deethylaiton
1.1
(FDR/Fdx)
7-Ethoxy-4trifluoromethylcoumarin
O-deethylaiton
4-Nitrophenol
3-hydroxylation
Simvastatin
6’βhydroxylation
Lovastatin
6’βhydroxylation
Atorvastatin
Resveratrol
4-hydroxylation
hydroxylation
Involved
human
P450
min-1
references
2E1
1.57
1A1
4.49
1A2
0.12
1A2
2.4
3A4
8.3
0.12
(PDR/Pdx)
2E1
33
Spatzenegger
et al. 2003
6.6
(H2O2)
3A4
3.1
Kim et al.
2011
13.6
(H2O2)
3A4
6.8
Kim et al.
2011
0.26
(H2O2)
3A4
19.6
Park et al.
2008
3A5
10.4
0.015
(t-BHP)
1A1
0.33
1A2
0.68
1B1
0.07
0.94
(PDR/Pdx)
Yamazaki et
al. 1996
Kim et al.
2002
Piver et al.
2004
Phenacetin
O-deethylation
<0.02
1A2
1.8
Yun et al.
2000
Chlorzoxazone
6-hydroxylation
0.21
(t-BHP)
2E1
5.78
Yamazaki et
al. 1996
157
8
158
159
Supplemental Figure 1. Chemical structures of substrates for CYP191A1 used in this study. The
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oxygenation sites by CYP191A1 were marked.
9
161
162
163
164
165
166
167
168
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Supplemental Figure 2. Amino acid sequence alignment of four P450s of CYP191A subfamily from
Mycobacterium genus. Amino acid sequences of CYPs 191A1 (from Mycobacterium smegmatis),
191A2 (from Mycobacterium avium subsp. Paratuberculosis), 191A3 (from Mycobacterium marinum
MM0399), and 191A4 (from Mycobacterium vanbaalenii PYR-1) were compared
(http://drnelson.uthsc.edu/CytochromeP450.html). This alignment is based on amino acid sequence
using Clustal W. When the amino acid sequence of CYP191A1 was compared to that of 191A2,
191A3, and 191A4, the identities of 191A1 to the corresponding CYP191A proteins were 69% 70%,
and 78%, respectively.
170
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171
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