Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Article abstract of DOI:10.1002/cbic.201800284

CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO³⁺), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H₂O₂, with optimum conversion surprisingly being achieved at ≈75 mm H₂O₂. More importantly, H₂O₂ tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H₂O₂. Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

Full text of DOI:10.1002/cbic.201800284

Accepted Article
Title: Effect of alternative redox partners and oxidizing agents in
CYP154C8 catalytic activity and product distribution
Authors: Bikash Dangi, Hyun Park, and Tae-Jin Oh
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201800284
Link to VoR: http://dx.doi.org/10.1002/cbic.201800284
A Journal of
www.chembiochem.org

ChemBioChem
10.1002/cbic.201800284
1
2
Effect of alternative redox partners and oxidizing agents in CYP154C8 catalytic activity
and product distribution
1
2,3
1,4,5,
3
Bikash Dangi , Hyun Park , Tae-Jin Oh
*
1
4
5
Department of Life Science and Biochemical Engineering, SunMoon University, 70 Sunmoon-ro
221, Tangjeong-myeon, Asan-si, Chungnam 31460, Republic of Korea
2
6
Unit of Polar Genomics, Korea Polar Research Institute, Incheon 21990, Republic of Korea
3
7
8
Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic
of Korea
4
9
0
Department of Pharmaceutical Engineering and Biotechnology, SunMoon University, 70
1
Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Republic of Korea
5
1
1
2
Genome-based BioIT Convergence Institute, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si,
1
Chungnam 31460, Republic of Korea
13
14
15
16
17
18
Author for correspondence: Prof. Tae-Jin Oh
Tel: +82(41)530-2677;
Fax: +82(41)530-2279;
E-mail: tjoh3782@sunmoon.ac.kr
Running title: Steroid hydroxylation of CYP154C8 depend on electron transport system
1
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
19
Abstract
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
CYP154C8 catalyzes the hydroxylation of diverse steroids as was previously demonstrated by
using an NADH-dependent system that included putidaredoxin and putidaredoxin reductase as
redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8
reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase, displayed an
altered catalytic activity compared with the NADH-dependent system. The NADPH-dependent
system showed multi-step oxidation of progesterone and other substrates including
androstenedione, testosterone and nandrolone. (Diacetoxyiodo)benzene is employed to generate
3
+
compound I (FeO ) actively supporting the redox reactions catalyzed by CYP154C8. In addition
to 16α-hydroxylation, progesterone and 11-ketoprogesterone were also hydroxylated at the
respective 6β position in the reaction supported by (diacetoxyiodo)benzene. CYP154C8 was
active in the presence of high concentration (>10 mM) of H
optimum conversion was achieved at ~75 mM H . More importantly, H
CYP154C8 showed a very low heme oxidation rate constant (K) even at high concentrations of
. Our results demonstrate that the alternative redox partner and oxidizing agents influence
2
O
2
, although surprisingly, the
2
O
2
2
O
2
tolerance of
H
2
O
2
the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly,
such choice affected the type and selectivity of the reaction catalyzed by the P450 enzyme.
36
3
7
8
Keywords: Cytochrome P450; (diacetoxyiodo)benzene; electron transport system; hydrogen
3
peroxide; steroid hydroxylation.
2
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
3
9
Introduction
Cytochrome P450s (CYPs) are widely distributed in all forms of life (archaea, bacteria and
eukarya) (1). Most CYP catalyzes the oxidative reaction utilizing O and two electrons supplied
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
2
by NAD(P)H and transferred via redox partners like flavin-containing reductase and iron-sulfur
ferredoxins (2). The mammalian CYPs are membrane bound and reduced by NADPH-
cytochrome P450 reductase (CPR), which is also a membrane-bound enzyme required for
electron transfer from NADPH to CYPs in the endoplasmic reticulum. The FAD- and FMN-
containing CPRs support CYPs of the class of II (3). The class I redox system includes bacterial
and mitochondrial iron-sulfur (Fe-S) containing proteins (ferredoxin/adrenodoxin, Fdx/Adx) and
FAD reductase (ferredoxin/adrenodoxin reductase, Fdr/Adr). In addition, flavin-dependent
proteins called flavodoxins (flavin mononucleotide containing proteins) are present in some
prokaryotic organisms and certain algae (4). A distinct group is built by the so-called self-
sufficient CYP in which a CPR-like reductase domain is fused to a monooxygenase domain (5-
11). One of the well-known forms is P450BM3 (CYP102A1) discovered in Bacillus megaterium,
a natural fusion of fatty acid hydroxylase P450 to a soluble CPR (5). In addition to CYPs that are
either linked to electron carrier proteins or require redox partner proteins to receive electrons
from reducing equivalent, a few unusual CYPs (CYP170A1, CYP154A1 and CYP170A1) have
been characterized with activity in the absence of redox partners (12-14). Only a few native
CYPs have been found to display peroxygenase and peroxidase functions, catalyzing the
peroxygenation of various substrates in the presence of H
20). Using the ‘peroxide shunt’ pathway, ferric CYP is directly moved into ferric hydroperoxo
intermediate known as compound 0. The H -shunt reaction is an attractive option for
2
2
O and other peroxy compounds (15-
2
O
2
monooxygenation reactions by CYP enzymes because these reactions are independent of redox
3
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
62
63
64
65
66
67
68
69
70
71
72
73
74
75
partner proteins and more importantly a low-cost of H
Many efforts have been made to develop artificial H
benefit as a practical biocatalyst (21-23). In addition, the H
2
O
2
can be significant on an industrial scale.
2
O
2
-dependent CYPs considering their
-dependent CYPs have been
2
O
2
deployed to oxidize non-native substrate in the presence of decoy molecules (20, 24-25). Further,
single oxygen donors (e.g., periodate and iodosobenzene) are also found to promote CYP-
catalyzed monooxygenation reactions, resulting in a highly reactive ferryl heme π-cation radical,
compound I (26-29). However, a few CYPs are known to display such characteristics in the
presence of specific chemical compounds. The characterization or synthetic application of CYPs
has often been mediated via one or more surrogate redox partners either in an isolated form or
after artificial fusion with CYP complex, due to the difficulty in obtaining native redox partners
(30-34). It is generally believed that the choice of the surrogate partners or their mode of action
does not affect the type and selectivity of the reactions catalyzed by CYPs (35). However, the
alternative redox partners might influence the catalytic efficiency and the product distribution
(29, 35-40).
76
77
78
79
80
81
82
83
84
CYPs like CYP106A1, CYP106A2, CYP109B1, CYP109E1, CYP154C3, CYP154C5,
CYP260A1 and CYP260B1 originated from the bacterial source are known to hydroxylate
steroids (32, 33, 41-45). CYP154C8 shows high similarity with CYP154C3 (74%) and
CYP154C5 (66%), both of which are reported to hydroxylate steroids at C16α position.
CYP154C8 was previously characterized with the NADH-dependent system comprising
putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) as redox partner proteins to transfer
electrons from NADH to CYP (46). It was found to hydroxylate diverse steroids at different
positions. Androstenedione, testosterone and 11-ketoprogesterone steroids were 16α-
hydroxylated and two other dihydroxylated products of progesterone were formed as a result of
4
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
85
86
87
88
89
90
91
92
subsequent hydroxylation of 16α-hydroxyprogesterone by CYP154C8. The major product of
corticosterone was found to be 21-hydroxycorticosterone, and substrate bearing hydroxyl or
carbonyl group at C11 and C21 position showed similar product formation with corticosterone.
In this work, the in vitro reactions with steroids (Scheme 1) mediated via NADPH-dependent
redox partner system as well as the oxidizing agents (diacetoxyiodo)benzene and H
2
2
O showed
changes in product distribution and catalytic activity of CYP154C8 compared with NADH-
dependent system. However, other oxidizing agents including sodium periodate, sodium chlorite
and tert-butyl hydroperoxide failed to support the in vitro reaction catalyzed by CYP154C8.
93
94
95
Materials and methods
Chemical and reagents
96
97
98
99
All steroid substrates were purchased from Tokyo Chemical Industry Co., Ltd. (Korea).
Isopropyl-1-thio-β- -galactopyranoside (IPTG), 1,4-dithiothreitol (DTT), and kanamycin were
D
obtained from Duchefa Bohemie (Korea). Ampicillin (Amp), chloramphenicol (Cm), α-
aminolevulinic acid (ALA), nicotinamide adenine dinucleotide (NADH), nicotinamide adenine
100
101
102
103
104
105
dinucleotide phosphate (NADPH), hydrogen peroxide (H
2
O ), (diacetoxyiodo)benzene,
2
cytochrome C, catalase, formate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose-6-
phosphate, spinach ferredoxin (Fdx) and spinach ferredoxin reductase (Fdr) were procured from
Sigma-Aldrich (Korea). Restriction enzymes were obtained from Takara Clontech (Korea). T4
DNA ligase, DNA polymerase, and dNTPs were supplied by Takara Bio (Japan). All other
chemicals were high-grade products obtained from commercially available sources.
106
5
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
107
Enzymatic in vitro assay with NADPH and its surrogate redox partner Fdx and Fdr
108
109
110
111
112
113
114
115
116
117
The heterologous expression and purification of CYP154C8 was performed as described
previously (46). Similarly, the surrogate redox partners, Pdx and Pdr, were expressed and
purified as described elsewhere (47). Reaction mixtures contained 3 µM substrate, 3 µM
CYP154C8, 6 µM Fdx, 0.1 U Fdr, 10 mM glucose-6-phosphate, 1 U glucose-6-phosphate
dehydrogenase, 100 µg/mL catalase, 1 mM MgCl and 250 µM NADPH. All the in vitro
2
reactions in the enzymatic assay were incubated at 30°C with shaking (200 rpm) for 2 h in 50
mM potassium phosphate buffer containing equal concentrations of substrate (500 µM) and CYP
enzyme (3 µM) in a final reaction volume of 0.5 mL. The reactions were extracted twice with
500 µL of ethyl acetate and dried under vacuum. The dried reaction mixture was dissolved in
HPLC solvent acetonitrile (70%) and water (30%) for further analysis.
1
18
19
1
Enzymatic in vitro assay with surrogate oxidant
120
121
122
123
124
125
126
127
The reactions were initiated separately with the addition of H
(diacetoxyidodo)benzene (exogenous oxidant). The in vitro activity of CYP154C8 was
optimized with different concentrations of H ranging from 0.2 ~ 100 mM, and 1 ~ 5 mM of
(diacetoxyidodo)benzene. The data shown here were obtained via in vitro reactions that included
75 mM H and 2 mM (diacetoxyidodo)benzene, separately. The reaction mixture (0.5 mL)
contained 500 µM steroid substrate and 3 µM CYP154C8 in potassium phosphate buffer (pH
7.4). Reactions were triggered by the addition of H and (diacetoxyidodo)benzene, separately
2
O
2
(biological hydroperoxide) and
2
O
2
2
O
2
2
O
2
at 30°C for 2 h. The reaction mixture was extracted as described previously (46).
128
6
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
129
Hydrogen peroxide tolerance of CYP154C8
130
131
132
133
134
135
136
137
The hydrogen peroxide tolerance of CYP154C8 was monitored using UV-visible spectroscopy
on a Biochrome Libra S35PC spectrophotometer. Oxidative modification of CYP154C8 (3 µM)
was carried out in the range of 0.2 ~ 100 mM concentrations of H
2
O
2
. After the addition of H
2
O
2,
the absorbance was recorded at a wavelength of 350 ~ 500 nm every 90 s for 30 min at room
temperature. The soret peak intensity (417 nm) of CYP154C8 was plotted against time, and the
data fitted using one phase decay in GraphPad Prism 6 software to calculate the rate constant for
heme oxidation (K). Associated absorbance amplitudes (A) for heme were calculated as the
difference between the highest and the lowest absorbance at soret peak.
1
38
39
1
Determination of catalytic efficiency and kinetic parameters
140
141
142
143
144
145
146
147
148
149
The time-dependent in vitro conversion of progesterone and 16α-hydroxyprogesterone was
carried out using 2 mM (diacetoxyidodo)benzene and 75 mM H
2
2
O , respectively. The reaction
mixture (500 µL) contained 1 µM CYP154C8 and 500 µM substrate. The reaction was
performed at 30°C with shaking (400 rpm) at intervals of 1 ~ 60 min. All the reaction mixture
was extracted as described previously, and the conversion (%) of each product at different time
intervals was calculated from the area of the product peaks based on HPLC chromatogram. The
product concentration with the substrate progesterone and 16α-hydroxyprogesterone in the
presence of (diacetoxyido)benzene and 75 mM H
2
2
O , respectively, was determined at different
substrate concentrations ranging from 25 ~ 400 µM. The reaction mixture was incubated and
extracted as described elsewhere. Assuming the absorbance properties of products and substrate
7
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
1
50
51
same, the products were quantified by correlating the peak area of the respective product(s) with
the combined peak area of product(s) and the substrate.
1
1
52
53
1
Product purification and characterization
154
155
156
157
158
159
160
161
162
Large-scale (300 mL) in vitro reactions were carried out to determine the structure of the
products catalyzed by CYP154C8. The reaction was carried out separately in a 15 mL volume to
determine the products obtained in the presence of 2 mM (diacetoxyidodo)benzene. All in vitro
reactions were carried out in 50 mM potassium phosphate buffer (pH 7.4) for 2 h in the presence
of 3 µM CYP154C8 and 500 µM substrate. The reaction mixture was extracted with 300 mL of
ethyl acetate and dried under reduced pressure, and was dissolved in HPLC-grade methanol. The
samples were filtered through 0.45-μm pore size PTFE filter and subjected to preparative HPLC
(Shimadzu) with a C18 column (Mightysil RP-18 GP, 150 × 4.6 mm, 5 µm, Kanto Chemical,
Japan) for the purification of the respective product peaks.
1
63
64
1
Analytical methods
165
166
167
168
169
170
171
The reaction mixture extracted after drying was used for analysis. The dried residue was
dissolved in acetonitrile for analysis by Ultra High Performance Liquid Chromatography
(UHPLC). The sample was injected into UHPLC and separated using Mightysil Reverse phase
C18 column (4.6 × 250 mm, 5 µm). Water (A) and acetonitrile (B) were used as mobile phases for
separation. The reaction mixture was analyzed using 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. Substrates and their products were detected by UV-A at 242 and 245 nm. All the
8
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
172
173
174
reaction mixtures were analyzed with SYNAPT G2-S/ACUITY UPLC liquid chromatography
quadrupole time-of-light/electrospray ionization mass spectrometry (Waters, Milford, MA, USA)
in positive ion mode.
175
176
177
178
179
180
The purified hydroxylated products were dissolved in dimethyl-sulfoxide (DMSO-d
6
) and
subjected to NMR analyses on 900 MHz using Varain Unity INOVA spectrometer (Varian, Palo
1
13
Alto, CA, USA). One-dimensional NMRs ( H-NMR and C-NMR) were performed by two-
dimensional NMR, heteronuclear multiple bond correlation, correlation spectroscopy, rotating-
frame overhauser effect spectroscopy (ROESY), and heteronuclear single quantum coherence
spectroscopy (HSQC) to delineate the structures when appropriate.
181
1
182
183
184
185
186
187
188
189
190
191
6β-Hydroxyprogesterone. H NMR (900 MHz, DMSO-d6): δ 5.67 (s, 1H), 5.13 (d, J = 2.9 Hz,
1H), 4.16 (q, J = 3.0 Hz, 1H), 2.59 (t, J = 9.2 Hz, 1H), 2.46 (ddd,J = 17.0, 15.0, 5.0 Hz, 1H),
2.21 (dt, J = 17.1, 3.5 Hz, 1H), 2.07 (s, 3H), 2.06 – 2.04 (m, 1H), 2.04 – 2.01 (m, 1H), 1.96 (ddd,
J =13.2, 5.1, 2.8 Hz, 1H), 1.86 (qd, J = 10.9, 3.3 Hz, 1H), 1.82 (dt, J = 13.6, 3.0 Hz, 1H), 1.65 (tt,
J = 12.4, 3.8 Hz, 1H), 1.61 (dd, J =14.1, 4.3 Hz, 1H), 1.60 – 1.58 (m, 0H), 1.58 (s, 1H), 1.46 –
1.38 (m, 2H), 1.29 (s, 3H), 1.20 – 1.16 (m, 1H), 1.16 – 1.13 (m, 2H), 0.91(td, J = 11.3, 4.2 Hz,
13
1H), 0.58 (s, 3H). C NMR (226 MHz, DMSO-d6): δ 209.08 (C20), 199.76 (C3), 169.40 (C5),
125.61 (C4), 71.46 (C6), 63.00 (C17), 55.66 (C14), 53.37 (C9), 43.85 (C13),39.11 (C7), 38.27
(C12), 38.02 (C10), 37.05 (C1), 34.33 (C2), 31.65 (C21), 29.82 (C8), 24.42 (C15), 22.71, 20.99
(C16), 19.39(C11), 13.50 (C18).
1
1
92
93
6β-Hydroxy,11-ketoprogesterone. H NMR (900 MHz, DMSO-d6): δ 5.69 (s, 1H), 5.24 (d, J =
1
2.9 Hz, 1H), 4.18 (q, J = 2.7 Hz, 1H), 2.82 (t, J = 9.2 Hz, 1H), 2.68 (d, J = 12.4Hz, 1H), 2.60
9
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
194
195
196
197
198
199
200
201
(ddd, J = 13.4, 5.0, 2.9 Hz, 1H), 2.51 – 2.48 (m, 1H), 2.46 (d, J = 12.4 Hz, 1H), 2.20 (qd, J = 11.3,
3.0 Hz, 1H), 2.17 (dt, J =17.2, 3.4 Hz, 1H), 2.12 (d, J = 11.4 Hz, 1H), 2.09 (dt, J = 13.2, 9.7 Hz,
1H), 2.06 (s, 3H), 1.91 (dt, J = 13.5, 2.9 Hz, 1H), 1.87 (td, J = 11.7,7.2 Hz, 1H), 1.82 – 1.75 (m,
1H), 1.80 – 1.73 (m, 1H), 1.62 (td, J = 14.2, 4.3 Hz, 1H), 1.48 (s, 3H), 1.42 (ddd, J = 13.5, 11.8,
13
3.2 Hz, 1H),1.26 (qd, J = 11.6, 10.9, 5.3 Hz, 1H), 0.52 (s, 2H). C NMR (226 MHz, DMSO-d6):
δ 209.61 (C11), 208.57 (C20), 199.88 (C3), 167.94 (C5), 126.16 (C4), 70.99 (C6), 61.43 (C9),
61.33 (C17),
56.17(C12), 53.49(C14), 46.71(C13), 39.32(C7), 37.78(C10), 35.79(C1),
34.06(C2), 31.43(C21), 31.02(C8), 23.93(C15), 23.27(C16), 18.92, 14.47(C18).
202
203
204
Results
Hydrogen peroxide tolerance of CYP154C8
205
206
207
208
209
210
211
212
213
Oxidative degradation of heme is a major challenge in H
determine the effect of H on CYP154C8 reaction, the H
range of 0.2 ~ 100 mM H . The decrease of soret absorbance in an oxidized form of
CYP154C8 was monitored at different concentrations of H at intervals up to 30 min. The
soret absorbance at different intervals was plotted against time. The soret peak intensity
decreased in time during exposure to > 1 mM H concentration (Figure 1). Surprisingly, the
heme oxidation rate constant (K) was low even at higher concentration of H (Table 1). These
tolerance even at high (>50 mM) H
2
O
2
-mediated CYP reaction. To
2
O
2
2
O
2
tolerance was analyzed in the
2
O
2
2
O
2
2
O
2
2
O
2
data demonstrate that CYP154C8 has high H
2
O
2
2
O
2
concentration.
2
14
15
2
Reaction with NADPH and surrogate redox partner Fdx and Fdr
10
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
In vitro reaction reconstituted with spinach surrogate redox partner, cofactor NADPH, glucose-6-
phosphate and glucose-6-phosphate dehydrogenase for cofactor regeneration as described in
Materials and Methods was highly preferred compared with NADH system and oxidizing agents.
The percentage of conversion for all substrates was found to be significantly increased with this
system compared with NADH-dependent system (data not shown). Androstenedione and
testosterone were completely converted into products. In addition to 16α-hydroxylated product
peaks, both the substrate reaction mixtures showed additional peaks in HPLC chromatogram,
respectively (Figure 2). LC-MS analysis of the reaction mixture revealed both additional peak as
dihydroxylated products of their respective substrates (Figure S1). These dihydroxylated
products must have resulted from subsequent hydroxylation of 16α-hydroxylated products in the
reaction. To confirm it, 16α-hydroxylated products of androstenedione and testosterone were
purified as described in Materials and Methods, and an in vitro reactions with 16α-
hydroxyandrostenedione and 16α-hydroxytesosterone were carried out instead of
androstenedione and testosterone, respectively. The HPLC (Figure S2) and LC-MS (data not
shown) analysis confirmed dihydroxylated products of androstenedione and testosterone which
were obtained as a result of sequential hydroxylation of 16α-hydroxyandrostenedione and 16α-
hydroxytestosterone, respectively. The nandrolone reaction mixture also showed multiple peaks
in HPLC chromatograms (Figure S3), where P4 was a major product while others yielded a very
low conversion. LC-MS analysis showed P1 as a dihydroxylated product, and the remaining 4
products were found to be hydroxylated at a single position (Figure S4). Androstenedione,
testosterone, and nandrolone were never converted to di-hydroxylated products when CYP154C8
was supported by the NADH-dependent system (46). 11-OH progesterone showed a possible
major peak of 16α-hydroxylation and a very low additional product peak (Figure S5, inset I).
11
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
239
240
241
242
243
244
245
246
247
248
249
Both the peaks were identified as monohydroxylated products of 11-hydroxyprogesterone.
However, 11-ketoprogesterone was hydroxylated to a single position (Figure S5, inset II). A
comparative HPLC and LC-MS analysis with the previous standard showed hydroxylation at
C16α-position (46). Progesterone reaction mixture supported by the NADPH-dependent system
showed similar product formation pattern compared with the NADH-dependent system based on
HPLC analysis (Figure S6, inset I). In our previous report, progesterone in the NADH-dependent
system was initially hydroxylated at a C16α position to form 16α-hydroxyprogesterone, which
was sequentially hydroxylated at two different positions to yield 6β,16α- dihydroxyprogesterone
and 2α,16α-dihydroxyprgestrone (46). The same 16α-hydroxyprogesterone and two
dihydroxylated products (6β,16α- dihydroxyprogesterone and 2α,16α-dihydroxyprgestrone) were
found to have formed with the NADPH-dependent system too (Figure S6, inset II).
2
50
51
2
CYP154C8 reaction with (diacetoxyiodo)benzene
252
253
254
255
256
257
258
259
260
261
In addition to the usual 16α-hydroxyprogesterone product (P1) from progesterone, another
monohydroxylated product (P2) was formed when the reaction was carried out with the surrogate
oxidant (diacetoxyiodo)benzene which was never observed with another system (Figure 3).
NMR-based structural elucidation of the product (P2) revealed as 6β-hydroxyprogesterone. A
very low yield of the two dihydroxylated products was detected in the same reaction mixture.
Further, when 16α-hydroxpogesterone was used as a substrate instead of progesterone supported
by (diacetoxyiodo)benzene, the reaction mixture showed the formation of two different
dihydroxylated products with the retention time matching precisely that of the two
dihydroxylated products in progesterone reaction mixture (Figure S7). Similarly, HPLC analysis
of 11-ketoprogesterone (Figure 4) and 11-hydroxyprogesterone (Figure S8) reaction mixture also
12
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
showed two monohydroxylated products, separately. LC-MS analysis (data not shown) revealed
both the peaks as monohydroxylated products of respective substrates. The structural elucidation
of P2 of 11-ketoprogesterone by NMR showed the hydroxylation at 6β-position, while another
product (P1) was identified as 16α-hydroxy,11-ketoprogesterone based on HPLC retention time
(Figure S9) and LC-MS (data not shown) comparison with an authentic standard 16α-
hydroxy,11-ketoprogesterone. The authentic standard was previously characterized by
CYP154C8 supported by the NADH-dependent system (46). A similar peak retention pattern of
11-hydroxyprogesterone was observed, suggesting that these products were also modified at
similar position found in 11-ketoprogesterone. Multiple peaks were observed in HPLC
chromatogram with testosterone, nandrolone and androstenedione reaction mixture (Figure S10).
LC-MS analysis of the reaction mixture showed at least four monohydroxylated products for
testosterone (Figure S11A) and nandrolone (Figure S11B) substrates and three
monohydroxylated products for androstenedione (Figure S11C) substrate. The major conversion
was related to 16α-hydroxylated product for androstenedione (Figure S12A) and testosterone
(Figure S12B) identified by HPLC and LC-MS (data not shown) compared with the authentic
standards. All other additional products obtained via reactions catalyzed by CYP154C8 were
found to be minor, and their selectivity was lower than that of the 16α-hydroxylated product.
Progesterone (~ 83%) was the most favored substrate, while all other substrates showed at least
35% conversion yield in the presence of (diacetoxyiodo)benzene (Table 2).
2
81
82
2
Reaction with H
2
O
2
2
83
84
CYP154C8 was active in the presence of high concentration (> 10 mM) of H
2
O . The optimum
2
2
conversion of the substrates occurred at ~75 mM H . Surprisingly, 16α-hydroxyprogesterone
2
O
2
13
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
285
286
287
288
289
290
291
292
293
294
295
296
was the most favored substrate for CYP154C8 in the presence of H
2
O
2
(Figure 5). Both products
containing 6β,16α- dihydroxyprogesterone and 2α,16α-dihydroxyprgestrone were predominantly
observed with an overall conversion of ~ 51 %, while the remaining substrates had a very low
conversion (Table S1). Although the percentage of conversion was too low for 11-
ketoprogesterone and 11-hydroxyprogesterone, both the substrate reactions showed two
monohydroxylated product peaks in HPLC (Figure S13), which were further confirmed by LC-
MS (data not shown). The retention times of 11-ketoprogesterone products were similar the
previously identified 6β-hydroxy,11-ketoprogesterone and 16α-hydroxy,11-ketoprogesterone
obtained in the reactions supported by H
2
2
O . The HPLC and LC-MS (data not shown)
comparison of progesterone (Figure S14A), androstenedione (Figure S14B) and testosterone
(Figure S14C) reaction mixture with the standard showed that the CYP154C8 in the presence of
H
2
2
O was region- and stereo-specific to 16α-hydroxylation.
2
97
98
2
Determination of catalytic efficiency and kinetic parameters
299
300
301
302
303
304
305
306
Progesterone was found to be the most favored substrate for CYP154C8 supported by
(diacetoxyiodo)benzene. In another reaction when 16α-hydroxyprogesterone was selected as a
substrate for CYP154C8 supported by H
2
2
O yielded a higher conversion compared with other
steroid substrates. Hence, progesterone and 16α-hydroxyprogesterone were selected to determine
the catalytic efficiency and kinetic parameters in the presence of (diacetoxyiodo)benzene and
H
2
O
2
, respectively. The K
m
and Kcat values for progesterone were estimated at 75.94 ± 10.64 µM
-1
and 2.32 ± 0.10 min , respectively (Figure 6A). Similarly, 16α-hydroxyprogesterone showed K
m
-1
and Kcat values of 134.50 ± 17.27 µM and 2.37 ± 0.13 min , respectively (Figure 6B). In
14
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
3
07
08
addition to the determination of the kinetic parameters, the time-dependent conversion of both
substrates has been demonstrated (Figure 7).
3
309
310
311
Discussion
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
The in vitro reaction with NADPH-dependent system was used to further characterize
CYP154C8. The catalytic efficiency of CYP154C8 was not only enhanced with NADPH-
dependent system compared with the NADH-dependent system, but also new products were
observed. The multi-step oxidation of progesterone by CYP154C8 with NADH-dependent
system has been previously been reported (46). The reaction supported by NADPH system
showed multi-step oxidation of progesterone, androstenedione, testosterone and nandrolone
indicating the preference for NADPH over NADH by CYP154C8. Such product formation in
NADPH system also showed the role of alternative redox partner and reducing equivalents in the
CYP154C8 catalytic efficiency and product distribution. In addition, it was also established that
the choice of appropriate reducing equivalents and surrogate redox partners might play an
important role in the catalytic efficiency and product distribution of CYP enzymes (29, 35-40,
48). The multi-step oxidation of steroids by CYP154C8 is novel by any bacterial source of CYPs,
although cytochrome P450BioI (CYP107H1) and MycG from bacterial source have already been
reported to catalyze the multi-step oxidation of fatty acids and mycinamycin-IV, respectively (35,
49). Some of the CYPs like CYP11A1, CYP17A1, CYP19A1 and CYP24A1 from the
mammalian source are widely known to catalyze the multi-step oxidation of steroids (50-53).
15
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
In addition to the 16α-hydroxylated products of the substrate progesterone and 11-
ketoprogesterone, the formation of 6β-hydroxylated products from the both substrates supported
by oxygen surrogate (diacetoxyiodo)benzene was unusual because such products were never
observed with NAD(P)H-dependent system, although a very low conversion occurred in the
presence of H
2
2
O (Scheme 2). However, such product formation in the presence of only oxygen
surrogates is not clear, allosteric effects of interactions with redox partners affect the CYP
activity and product distribution. Recently, it was reported that CYP17A1 yields different
products in the presence of iodosobenzene and NADPH-P450 reductase, to form compound I
(29). The effect of cytochrome b5 on the catalytic activity and product formation of CYPs has
been widely studied, and shown to induce the activity of specific CYPs (54, 55). In another
experiment, MycG, a CYP derived from bacterial source showed altered catalytic type in
conjunction with an alternate surrogate redox partner, which highlights the role of variant redox
partner in protein-protein interaction in the catalytic activity of CYP enzyme (35). In addition to
electron transfer, the complex of CYP101A1 with its natural redox partner protein, Pdx
demonstrates an important allosteric regulatory role of these redox partner proteins bound with
CYP (56, 57). Few CYPs are known to possess activity in the presence of
(diacetoxyiodo)benzene. Mammalian and bacterial CYPs such as CYP5A1, CYP17A1, CYP121,
CYP101A1 (P450cam) and CYP106A2 are known to show catalytic activity in the presence of
iodobenzene as a single oxygen donor (30, 58-61). The oxygenation mechanism supported by
(diacetoxyiodo)benzene might be similar to iodosobenzene, probably mediated via a two-
electron transfer of a single oxygen atom from oxidant to ferric CYP to generate compound I,
which is involved in substrate monoxygenation (58). CYP154C8 hydroxylation occurred usually
at the α-face of D-ring to steroid substrates. Interestingly, the hydroxylation occurred at the β-
16
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
face of progesterone and 11-ketoprogesterone and regioselectivity switched from D-ring to B-
ring. As reported previously, the 16α-hydroxyprogesterone was sequentially hydroxylated at 2α
and 6β position to yield the respective dihydroxylated product of progesterone (46). CYP17A1
unusual to its function hydroxylated at 6β-position when 16α,17α-dihydroxyprogesterone was
used as a substrate, which was attributed due to the presence of two hydroxyl groups on the 16α-
and 17α-position (29). The switch in hydroxylation from D-ring to B-ring by CYP154C8 in
presence of (diacetoxyiodo)benzene and H
2
2
O indicates the effect of oxidation system. Although,
NADPH as well as NADH-dependent system efficiently supported the CYP154C8 activity, they
never induced formation of 6β-hydroxylated product from the substrate progesterone and 11-
ketoprogesterone or/with any other substrates, suggesting the role of a redox partner in protein-
protein interactions for modulating the specificity of CYP. In addition, the functional groups
such as hydroxyl or the carbonyl group in the substrate may also influence the selectivity of
hydroxylation, although such switch in selectivity of different CYPs may not be the same (35,
39). The B-ring hydroxylation by CYP154C8 supported by (diacetoxyiodo)benzene and H
2
2
O is
unclear. However, the hydroxylation at C16α-position of progesterone might play a key role in
subsequent hydroxylation to A-ring and B-ring yielding 2α,16α-dihydroxyprogesterone and
6β,16α-dihydroxyprogesterone, respectively.
368
369
370
371
372
373
The peroxide shunt pathway uses H
2
O (or the oxygen donors) to drive CYPs, and
2
represents one of the efficient ways of using these enzymes for industrial application. However,
the oxidative degradation of heme by peroxide has been a major issue (15, 62). The activity of
CYP154C8 in the presence of high concentration of H
CYP154C8 displayed comparative tolerance to H , although the in vitro activity was only
observed with > 10 mM H . More importantly, the heme oxidation rate constant (K) of
2
O
2
is interesting and surprising.
2
O
2
2
O
2
17
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
-
1
374
375
376
377
378
379
380
381
382
383
384
385
386
CYP154C8 even at high concentration (100 mM) of H
A widely studied CYP, CYP152L1 uses H to catalyze reactions and the recent studies
supporting its hydrogen peroxide tolerance showed greater H tolerance than other CYPs
2
O
2
was consistently low (K > 0.6 min ).
2
O
2
2
O
2
(CYP121A1, P450 BM3 and CYP51B1) (63). The comparison of previously reported heme
oxidation rate constant (K) of CYP152L1 and CYP154C8 showed a higher tolerance of
CYP154C8 to H
2
O
2
for 30 min, while other CYPs were incubated for 1 h with different
. The activity of CYP154C8 in the presence of H with steroid
concentrations of H
2
O
2
2
O
2
substrates was low; in contrast, the elevated activity towards 16α-hydroxyprogesterone was
unusual. 6β-hydroxyprogesterone and 16α-hydroxyprogesterone are pharmaceutically important
compounds. 6β-hydroxyprogesterone is used as an intermediate for the synthesis of anticancer
compound 6β,14α-dihydroxyandrost-4-ene-3,17-dione, an inhibitor of the growth of breast
cancer cells and an inhibitor of the 5α-reductase activity in male rats, representing a potential
lead for the development of drugs against prostate cancer (64-67).
387
388
389
390
391
392
393
In conclusion, CYP154C8 catalyzes the sequential oxidation of steroid substrates that prefers
NADPH over NADH-dependent system. In addition, the use of alternative surrogate redox
partners and reducing equivalents might alter the catalytic efficiency and product distribution.
The unexpected change in product distribution pattern of steroid substrates observed with
NADPH, (diacetoxyiodo)benzene and H
2
2
O systems indicates the altered role of active oxygen
species in P450-mediated oxidation reactions. The optimum activity of CYP154C8 in the
presence of high concentrations of H
2
2
O is unusual and warrants further studies.
3
94
95
3
Acknowledgments
18
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
396
397
398
399
400
401
This research was supported by a grant (NRF-2016R1D1A3B03933814) of the Basic Science
Research Program through the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology, Republic of Korea. In addition, this work was
supported by the Korea Polar Research Institute (grant no. PE18210). We would like to thank
Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chungbuk, Korea for
NMR analyses.
402
4
03
04
Conflict of interest
4
The authors declare that they have no conflict of interest.
405
406
407
References
408
409
410
411
412
1. http://www.drnelson.uthsc.edu/cytochromeP450.html.
2. F.P. Guengerich, A.W. Munro, J. Biol. Chem. 2013, 288, 17065-17073.
3. A.V. Pandey, C.E. Flück, Pharmacol. Ther. 2013, 138, 229-254.
4. K.J. McLean, D. Luciakova, J. Belcher, K.L. Tee, A.W. Munro, Adv. Exp. Med. Biol.
2015, 851, 299-317.
4
13
14
5. L.O. Narhi, A.J. Fulco, J. Biol. Chem. 1986, 261, 7160-7169.
6. R.D. Mot, A.H. Parret, Trends Microbiol. 2002, 10, 502-508.
4
19
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
415
416
417
418
419
420
421
422
423
424
425
7. A.J. Warman, J.W. Robinson, D. Luciakova, A.D. Lawrence, K.R. Marshall, M.J.
Warren, M.R. Cheesman, S.E. Rigby, A.W. Munro, K.J. McLean, FEBS J. 2012, 279,
1675-1693.
8. M. Tavanti, J.L. Porter, S. Sabatini, N.J. Turner, S.L. Flitsch, ChemCatChem 2018, 10,
1042.
9. D. Minerdi, S.J. Sadeghi, G.D. Nardo, F. Rua, P. Castignano, G. Gilardi, Mol. Microbiol.
2015, 95, 539-554.
10. Y. Yin, H. Yu, Z. Luan, R. Li, P. Ouyang, J. Liu, J. Xu, ChemBioChem 2014, 15, 2443-
2449.
11. J.L. Porter, J. Manning, S. Sabatini, M. Tavanti, N.J. Turner, S.L. Flitsch,
ChemBioChem 2018, 19, 513.
426
427
428
429
430
431
432
433
434
435
436
437
12. R. Bernhardt, J. Biotechnol. 2006, 124, 128-145.
13. B. Zhao, L. Lei, D.G. Vassylyev, X. Lin, D.E. Cane, S.L. Kelly, H. Yuan, D.C. Lamb,
M.R. Waterman, J. Biol. Chem. 2009, 284, 36711-36719.
14. Q. Cheng, D.C. Lamb, S.L. Kelly, L. Lei, F.P. Guengerich, J. Am. Chem. Soc. 2010, 132,
15173-15175.
15. P.R. Ortiz de Montellano, Chem. Rev. 2010, 110, 932-948.
16. I. Matsunaga, A. Ueda, T. Sumimoto, K. Ichihara, M. Ayata, H. Ogura, Arch. Biochem.
Biophys. 2001, 394, 45-53.
17. M. Girhard, S. Schuster, M. Dietrich, P. Dürre, V.B. Urlacher, Biochem. Biophys. Res.
Commun. 2007, 362, 114-119.
18. M.A. Rude, T.S Baron, S. Brubaker, M. Alibhai, S.B. Del Cardayre, A. Schirmer, Appl.
Environ. Microbiol. 2011, 77, 1718-1727.
20
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
438
439
440
441
442
19. I. Matsunaga, M. Yamada, E. Kusunone, T. Miki, K. Ichihara, J. Biochem. 1998, 124,
105-110.
20. O. Shoji, T. Fujishiro, H. Nakajima, M. Kim, S. Nagano, Y. Shiro, Y. Watanabe, Angew.
Chem. Int. Ed. 2007, 46, 3656-3659.
21. P.C. Cirino, F.H. Arnold, Angew. Chem. Int. Ed. Engl. 2003, 42, 3299-3301.
443
444
445
446
447
22. S. Kumar, C. Chen, D. Waxman, J. Biol. Chem. 2005, 280, 19569-19575.
23. R.K. Behera, S. Goyal, S. Mazumdar, J. Inorg. Biochem. 2010, 104, 1185-1194.
24. N. Ma, Z. Chen, J. Chen, J. Chen, C. Wang, H. Zhou, L. Yao, O. Shoji, Y. Watanabe, Z.
Cong, Angew. Chem. Int. Ed. Engl. 2018, 57, 7628.
25. H. Onoda, O. Shoji, Y. Watanabe, Dalton Trans. 2015, 44, 15316-15323.
448
449
450
451
452
453
454
26. A. Berg, K. Carlstrom, J.-A. Gustafsson, M. Ingelman-Sundberg, Biochem. Biophys. Res.
Commun. 1975, 66, 1414-1423.
27. J.-A. Gustafsson, J. Bergman, FEBS Lett. 1976, 70, 276-280.
28. F. Lichtenberger, W. Nastainczyk, V. Ullrich, Biochem. Biophys. Res. Commun. 1976,
70, 939-946.
29. F.K. Yoshimoto, E. Gonzalez, R.J. Auchus, and F.P. Guengerich, J. Biol. Chem. 2016,
291, 17143-17164.
455
456
457
458
459
30. J.K. Kulig, C. Spandolf, R. Hyde, A.C. Ruzzini, L.D. Eltis, G. Gronberg, M.A. Hayes, G.
Grogan, Bioorg. Med. Chem. 2015, 23, 5603-5609.
31. K.J. McLean, M. Hans, B. Meijrink, W.B. van Scheppingen, A. Vollebregt, K.L. Tee,
J.M van der Laan, D. Leys, A.W. Munro, M.A. van den Berg, Proc. Natl. Acad. Sci.
2015, 112, 2847-2852.
21
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
460
461
462
463
464
32. T. Makino, Y. Katsuyama, T. Otomatsu, N. Misawa, Y. Ohnishi, Appl. Environ.
Microbiol. 2014, 80, 1371-1379.
33. Y. Khatri, M. Ringle., M. Lisurek, J.P. von Kries, J. Zapp, R. Bernhardt, ChemBioChem
2016, 17, 90.
34. R. Bernhardt, V.B. Urlacher, Appl. Microbiol. Biotechnol. 2014, 98, 6185-6203.
465
466
467
468
469
470
471
472
35. W. Zhang, Y. Liu, J. Yan, S. Cao, F. Bai, Y. Yang, S. Huang, L. Yao, Y. Anzai, F. Kato,
L.M. Podust, D.H. Sherman, S. Li, J. Am. Chem. Soc. 2014, 136, 3640-3646.
36. R.C. Tuckey, K.J. Cameron, Biochim. Biophys. Acta. 1993, 1163, 185-194.
37. Y. Khatri, A. Schifrin, R. Bernhardt, FEBS Lett. 2017, 591, 1126-1140.
38. S.G. Bell, A. Dale, N.H. Rees, L.L. Wong, Appl. Microbiol. Biotechnol. 2010, 86, 163-
175.
39. E. Jung, B.G. Park, M.M. Ahsan, J. Kim, H. Yun, K.Y. Choi, B.G. Kim, Appl.
Microbiol. Biotechnol. 2016, 100, 10375-10384.
4
73
74
40. W. Yang, S.G. Bell, H. Wang, W. Zhou, N. Hoskins, A. Dale, M. Bartiam, L.L. Wong,
4
Z. Rao, J. Biol. Chem. 2010, 285, 27372-27384.
475
476
477
478
479
480
481
482
41. F.M. Kiss, D. Schmitz, J. Zapp, Appl Microbiol Biotechnol. 2015, 99, 8495.
42. H. Agematu, N. Matsumoto, Y. Fujii, H. Kabumoto, S. Doi, K. Machida, J. Ishikawa, A.
Arisawa, Biosci. Biotechnol. Biochem. 2006, 70, 307-311.
43. I.K. Jóźwik, F.M. Kiss, Ł. Gricman, A. Abdulmughni, E. Brill, J. Zapp, J. Pleiss, R.
Bernhardt, A.W. Thunnissen, FEBS J. 2016, 283, 4128-4148.
44. P. Bracco, D.B. Janssen, A. Schallmey, Microb. Cell Fact. 2013, 12, 95.
45. S.G. Salamanca-Pinzon, Y. Khatri, Y. Carius, L. Keller, R. Müller, C.R. Lancaster, R.
Bernhardt, FEBS Lett. 2016, 590, 1838-1851.
22
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
46. B. Dangi, K.H. Kim, S.H. Kang, T.J. Oh. ChemBioChem 2018, 19, 1066-1077.
47. S. Bhattarai, K. Liou, T.J. Oh, Arch. Biochem. Biophys. 2013, 539, 63-69.
48. Y. Anzai, S. Li, R.M. Chaulagain, K. Kinoshita, F. Kato, J. Montgomery, D.H. Sherman,
Chem. Biol. 2008, 15, 950.
49. M.J. Cryle, J.J. De Voss, Chem. Commun, 2004, 86-87.
50. R. Davydov, N. Strushkevich, D. Smil, A. Yantsevich, A. Gilep, S. Usanov, B.M.
Hoffman, Biochemistry 2015, 54, 7089-7097.
51. J.R. Berrie, R.A. Williams, K.E. Smith, J. Steroid Biochem. Mol. Biol. 1999, 71, 153-
165.
52. Y. Osawa, K. Shibata, D. Rohrer, C. Weeks, W.L. Duax, J. Am. Chem. Soc. 1975, 97,
4400-4402.
53. M.J. Beckman, P. Tadikonda, E. Werner, J. Prahl, S. Yamada, H.F. DeLUCA
Biochemistry. 1996, 35, 8645-8472.
54. L. Waskell. J.-J.P. Kim, (Ortiz de Montellano, P. R., ed) pp. 33-60, Springer, New York,
2015.
55. M.R. Bhatt, Y. Khatri, R.J. Rodgers, L.L Martin, J. Steroid Biochem. Mol. Biol. 2017,
170, 2-18.
56. Y. Hiruma, M.A. Hass, Y. Kikui, W.M. Liu, B. Olmez, S.P. Skinner, A. Blok, A.
Kloosterman, H. Koteishi, F. Lohr, H. Schwalbe, M. Nojiri, M. Ubbink, J. Mol. Biol.
2013, 425, 4353-4365.
57. S. Tripathi, H. Li, T.L. Poulos, Science 2013, 340, 1227-1230.
58. E.G. Hrycay, S.M. Bandiera, Arch. Biochem. Biophys. 2012, 522, 71-89.
23
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
505
506
507
508
509
510
511
512
59. K. Dornevil, I. Davis, A.J. Fielding, J.R. Terrell, L. Ma, A. Liu, J. Biol. Chem. 2017,
292, 13645-13657.
60. M.H. Gelb, D.C. Heimbrook, P. Mälkönen, S.G. Sligar, Biochemistry 1982, 21, 370-377.
61. A. Berg, M. Ingelman-Sundberg, J.A. Gustafsson, J. Biol. Chem. 1979, 254, 5264-5271.
62. A.W. Munro, H.M. Girvan, A.E. Mason, A.J Dunford, K.J McLean, Trends Biochem.
Sci. 2013, 38, 140-150.
63. S. Matthews, K.L. Tee, N.J. Rattray, K.J. McLean, D. Leys, D.A. Parker, R.T. Blankley,
A.W. Munro, FEBS Lett. 2017, 591, 737-750.
513
514
515
516
517
518
64. U. Raju, H.L. Bradlow, M. Levitz, Ann. N. Y. Acad. Sci. 1990, 586, 83-87.
65. J. Marcos, W.Y. Craig, G.E. Palomaki, E.M. Kloza, J.E. Haddow, M. Roberson, L.A.
Bradley, C.H. Shackleton, Prenat. Diagn. 2009, 29, 771-780.
66. A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, K. Uematsu, J. Biol.
Chem. 2001, 276, 4604-4610.
67. K.E. Smith, S. Latif, D.N. Kirk, J. Steroid Biochem. 1990, 35, 115-120.
24
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
519
520
521
522
Table 1. Rates of heme oxidation constant (K) and absorbance amplitude (A) for heme soret peak
absorbance change at 417 nm of CYP154C8. Data were fitted in one-phase exponential decay
using GraphPad Prism 6. ND denotes “not determined” due to small amplitudes of heme
absorbance change.
523
25
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
524
525
526
527
528
529
530
Table 2. The conversion percentage of steroid substrates and selectivity of product formation
supported by (diacetoxyiodo)benzene. The table also shows the retention times of substrate and
their respective products. The product quantification was performed by correlating the peak area
of the products with the combined peak area of products and substrate. 16-OHP, 16α-
hydroxyprogesterone; 6-OHP, 6β-hydroxyprogesterone; 16-OHA, 16α-hydroxyandrostenedione;
and 16-OHT, 16α-hydroxyandrostenedione. P1-P4 represent the different products formed in the
reactions with respective substrates.
Substrates
Conversion (%)
Selectivity (%)
Retention time (min)
Progesterone
83.3
16α-OHP (70.0), 6β-OHP
S (26.6), 16-OHP (14.6), 6-OHP
(16.8)
(25.0)
Androstenedione
Testosterone
Nandrolone
36.2
37.5
68.6
16α-OHA (66.0), P1 (9.4), S (18.7), 16-OHA (13.7), P1 (13.2),
P2 (9.5), P3 (15.0)
P2 (13.9), P3 (14.7)
16α-OHT (76.0), P1 (3.0),
P2 (17.0), P3 (4.0)
S (17.2), 16α-OHT (12.1), P1 (11.2),
P2 (11.9), P3 (13.8)
P1 (13.5), P2 (31.5), P3
S (16.0), P1 (10.2), P2 (10.8), P3
(11.2), P4 (13.2)
(50.4) P4 (4.3)
1
1
1
1-Hydroxyprogesterone
1-Ketoprogesterone
69.0
50.3
14.0
P1 (61.8) P2 (38.2)
P1 (75.0), P2 (25.0)
P1 (9.5), P2 (4.5)
S (15.6), P1 (10.7), P2 (11.0)
S (17.7), P1 (12.5), P2 (13.0)
S (14.6), P1 (11.3), P2 (12.0)
6α-Hydroxyprogesterone
531
26
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
532
Scheme 1. Steroids used as substrates for further characterization of CYP154C8.
533
27
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
5
34
35
Scheme 2. Hydroxylation of progesterone (A) and 11-ketoprogesterone (B) mediated by
5
CYP154C8.
536
28
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201800284
537
538
539
Figure 1. H
2
O
2
tolerance by CYP154C8. The spectra show the absorbance for 30 min at the
interval of 1.5 min at H
2
O concentration. A, 1 mM; B, 3 mM; C, 5 mM; D, 20 mM; E, 50 mM;
2
and F, 100 mM.
5
40
41
5
29
This article is protected by copyright. All rights reserved.