Acetate anion-triggered peroxygenation of non-native substrates by wild-type cytochrome P450s

Article abstract of DOI:10.1039/c5dt00797f

Cytochrome P450_SPα (P450_SPα) and cytochrome P450_BSβ (P450_BSβ) belonging to the CYP152 family of enzymes (CYP152s) can utilize H₂O₂ efficiently as an oxidant for the generation of compound I. Although P450_SPα and P450_BSβ have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as "decoy molecules." Although we had assumed that acetic acid did not serve as a decoy molecule, P450_SPα and P450_BSβ efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450_BSβ in the presence of 1 M acetate anion reached 590 ± 30 min^-1.

Full text of DOI:10.1039/c5dt00797f

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Acetate anion-triggered peroxygenation of non-
native substrates by wild-type cytochrome P450s†
Cite this: DOI: 10.1039/c5dt00797f
Hiroki Onoda,a Osami Shoji*a and Yoshihito Watanabe*a,b
Cytochrome P450SPα (P450SPα) and cytochrome P450BSβ (P450BSβ) belonging to the CYP152 family of
enzymes (CYP152s) can utilize H2O2 efficiently as an oxidant for the generation of compound I. Although
P450SPα and P450BSβ have very high substrate specificity and catalyse hydroxylation of long-chain fatty
acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including
medium chain length n-alkyl carboxylic acids as “decoy molecules.” Although we had assumed that
acetic acid did not serve as a decoy molecule, P450SPα and P450BSβ efficiently catalysed oxidation of
non-native substrates when the reaction was carried out at a high concentration of acetate anion. The
turnover rate for epoxidation of styrene catalysed by P450BSβ in the presence of 1 M acetate anion
Received 24th February 2015,
Accepted 29th May 2015
DOI: 10.1039/c5dt00797f
www.rsc.org/dalton
reached 590 30 min−1
.
Introduction
Cytochrome P450s (P450s or CYPs) are heme–thiolate enzymes
that catalyse the monooxygenation of less reactive substrates
using molecular oxygen.1 Given that P450s can be obtained in
the soluble form by using typical Escherichia coli gene-
expression systems,2–5 such systems have been regarded as
potential candidates for the development of biocatalysts. In
general, P450s consume a stoichiometric amount of an expens-
ive cofactor (NAD(P)H) for the reductive activation of molecular
oxygen to generate an active species (compound I: oxoferryl
porphyrin π-cation radical) (Scheme 1).6 In contrast, P450BSβ
(CYP152A1),7 P450SPα (CYP152B1)8 and P450CLA (CYP152A2),9
which belong to the CYP152 family of enzymes, efficiently
utilize hydrogen peroxide (peroxide-shunt pathway)10 instead
of molecular oxygen and electrons from NAD(P)H. Given the
low cost of hydrogen peroxide, the CYP152 family of enzymes
constitutes good candidates as biocatalysts for organic syn-
thesis. However, the substrate specificity of the CYP152 family
of enzymes is very high; indeed, these enzymes catalyse the
oxidation of long-chain fatty acids exclusively.8 The catalytic
reaction mechanism was proposed based on the crystal struc-
Scheme 1 The catalytic cycle of P450s, including the hydrogen per-
oxide-shunt pathway.
aDepartment of Chemistry, Graduate School of Science, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya 464-8602, Japan. E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp,
p47297a@nucc.cc.nagoya-u.ac.jp
11
ture analysis of the palmitic acid bound form of P450BSβ
(Scheme 2A, Fig. 1). The terminal carboxylate of the long-chain
fatty acid interacts with Arg-242 at the distal side of heme and
serves as a general acid–base catalyst in the generation of com-
pound I using hydrogen peroxide. This carboxylate–Arg salt
bridge is very important for the utilization of hydrogen per-
oxide. In fact, a similar carboxylate–Arg salt bridge is observed
bResearch Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan
†Electronic supplementary information (ESI) available: Active site structures of
AaeUPO and AauDyP, gas chromatography of the product mixture after oxidation
of styrene by P450 in the presence of 1 M acetate anion. See DOI: 10.1039/
c5dt00797f
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Scheme 2 (A) Proposed catalytic hydroxylation mechanism for hydroxylation of palmitic acid catalysed by P450BSβ, in which the carboxylate of pal-
mitic acid interacts with Arg-242 to serve as a general acid–base catalyst for the generation of compound I (oxoferryl porphyrin π-cation radicals)
using hydrogen peroxide as an oxidant. (B) Proposed catalytic hydroxylation mechanism for epoxidation of styrene catalysed by P450BSβ in the pres-
ence of heptanoic acid as a decoy molecule. The carboxylate of heptanoic acid interacts with Arg-242 and serves as a general acid–base catalyst.
in a peroxygenase from Agrocybe aegerita (AaeUPO)12 and a dye- chain length constitute the best decoy molecules to enable
decolourizing peroxidase from Auricularia auricula-judae non-natural substrate oxidation by P450BSβ. However, although
(AauDyP)13 (Fig. S1†). P450BSβ and P450SPα cannot oxidize non- the effect of carboxylic acids with a short alkyl chain on the
native substrates efficiently because the terminal carboxylate reactivity was lower than those with a medium alkyl chain, it is
of the long-chain fatty acid is involved in the formation of possible to increase the concentration of carboxylic acids with
compound I. Recently, we have reported that P450BSβ and a short alkyl chain significantly because of their high solubility
P450SPα can catalyse the oxidation of non-native substrates in aqueous solution. Acetic acid is the simplest alkyl carboxylic
such as guaiacol,14 thioanisole,15 styrene,16 ethylbenzene14 acid and is miscible in all proportions with water. If acetic acid
and 1-methoxynaphthalene17 in the presence of n-alkyl car- could serve as a decoy molecule and the active species was gen-
boxylic acids with a medium chain length such as heptanoic erated, we reasoned that it would be possible to perform the
and hexanoic acid, which act as so-called “decoy molecules” oxidation of various non-native substrates by using wild-type
(Scheme 2B). Given that the inclusion of 20 mM acetic or pro- P450SPα and P450BSβ by simply increasing the concentration of
pionic acid did not accelerate the one-electron oxidation of the acetate anion in the reaction buffer. Herein, we report a
guaiacol catalysed by P450BSβ in the first screening of decoy simple approach to perform non-native substrate oxidation by
molecules,14 we had assumed that carboxylic acids bearing a using wild-type P450SPα and P450BSβ by replacing the reaction
very short alkyl chain were not suitable as decoy molecules14 buffer with the one containing a high concentration of acetic
and we concluded that n-alkyl carboxylic acids with a medium acid (acetate anion).
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Fig. 1 Overall structure (left) and active-site structure (right) of the palmitic acid-bound form of P450BSβ (A, PDB code: 1IZO) and P450SPα (B, PDB
code: 3AWM). The heme and palmitic acids are represented as stick models. The key amino acid residues in the active site of P450BSβ (Arg-242 and
Cys-363) and P450SPα (Arg-241 and Cys-361) are represented as stick models.
dine tag at its N-terminal19 were expressed in Escherichia coli
M15 (pREP4) and purified by nickel chelate affinity chromato-
Materials and methods
Materials
graphy (HiTrap, GE Healthcare Bio-Sciences, USA). Four P450s
All chemical reagents were purchased from commercial were further purified by gel filtration on a HiPrep 16/60 Sepha-
sources and used without further purification except for cryl S200 HR column (GE Healthcare Bio-Sciences) using
styrene. The following chemicals were purchased from Nacalai BioAssist eZ (TOSOH, Japan) according to a reported pro-
Tesque Inc. (Kyoto, Japan): styrene oxide, (R)-styrene oxide, cedure.19 The purity of P450s was estimated by SDS-PAGE and
propionic acid, hexanoic acid, octanoic acid, sodium acetate, the concentration of P450s was determined by using CO differ-
sodium carbonate, potassium dihydrogen phosphate, dipotas- ence spectra.2,20
sium phosphate, potassium chloride, glycerol, dichloro-
Measurements
methane and chloroform. Hydrogen peroxide, guaiacol,
1-methoxynaphthalene, acetic acid and heptanoic acid were
obtained from WAKO Pure Chemical Industries, Ltd (Osaka,
Japan). Butyric acid, pentanoic acid and styrene were obtained
from Tokyo Chemical Industry Co. (Tokyo, Japan). 2-Phenyl-
2-propanol and phenylacetaldehyde were obtained from
Sigma-Aldrich Co., (St. Louis, MO).
UV-Vis spectra were recorded with Shimadzu UV-2600 and
UV-2500 PC spectrophotometers. CD spectroscopy was per-
formed with a JASCO CORPORATION J-720WN. Gas chromato-
graphy (GC) analysis was performed with a Shimadzu GC-2014
ATF equipped with a cyclosil-β column (Agilent Technologies,
Inc., 30 m × 0.25 mm).
Preparation of recombinant forms of P450SPα and P450BSβ
Oxidation of 1-methoxynaphthalene
The recombinant P450SPα and its F288G mutant with a GST The oxidation of 1-methoxynaphthalene was carried out in
tag at its N-terminal18 were expressed in Escherichia coli BL21 0.1 M potassium phosphate buffer (pH 7.0) in the presence of
and purified by GST affinity chromatography (Glutathione 0.5 μM P450 and 0.65 mM 1-methoxynaphthalene. 1-Methoxy-
Sepharose 4 Fast Flow, GE Healthcare Bio-Sciences, USA). The naphthalene was added as an ethanol solution. To the reaction
recombinant P450BSβ and its G290F mutant with a 6 × histi- mixture, 5 mM carboxylic acid or 5–500 mM sodium acetate
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was added as a decoy molecule. If necessary, the pH was using an authentic sample of (R)-styrene oxide. The conditions
adjusted to 7.0 with 2 M HCl. The reaction was initiated by the for gas chromatography were as follows: column temperature:
addition of 100 mM hydrogen peroxide (final concentration 90 °C (35 min); 10 °C min−1; 190 °C (20 min), injection tem-
5 mM). The total volume of the reaction mixture was 1 mL, perature: 250 °C, interface temperature: 250 °C, detector tem-
including 5% ethanol. The reaction was performed at 25 °C for perature: 250 °C, carrier gas: helium. The retention times of
1 min and the resulting reaction mixture was extracted with styrene, products and the internal standard were as follows:
1 mL chloroform. The turnover rate for the formation of styrene (3.6 min), (R)-styrene oxide (13.3 min), (S)-styrene oxide
Russig’s blue was estimated from the absorption at 634.5 nm (14.0 min), phenylacetaldehyde (15.6 min) and 2-phenyl-2-pro-
with a molar absorption coefficient of ε = 1.8 × 104 M−1 cm−1 panol (33.4 min). All values are the average of at least three
in chloroform according to a reported method.17 All values are measurements and are expressed in units of nmol product per
the average of at least three measurements and are expressed min per nmol P450.21
in units of nmol product per min per nmol P450.21
Oxidation of styrene
Results and discussion
Styrene was purified by aluminum column chromatography to
remove the polymerization inhibitor and used immediately The hydroxylation of 1-methoxynaphthalene catalysed by
after the purification. An ethanol solution of styrene (100 mM) P450SPα in the presence of different concentrations
was prepared as the stock solution and kept on ice under pro- (0–500 mM) of acetic acid (acetate anion) was performed to
tection from light. The reaction was carried out in 0.1 M pot- examine whether acetic acid serves as a decoy molecule
assium phosphate buffer (pH 7.0) at 25 °C for 1 min in the (Fig. 2). We selected 1-methoxynaphthalene as a substrate
presence of 1 μM P450, 4 mM styrene and 20–1000 mM because the catalytic activity of aromatic hydroxylation can be
sodium acetate. The pH was adjusted to 7.0 with 2 M HCl. The estimated by monitoring the colour change of the reaction
reaction was initiated by the addition of 100 mM hydrogen mixture due to
a subsequent dimerization reaction of
peroxide (final concentration 4 mM). The total volume of the 4-methoxy-1-naphthol giving Russig’s blue.17 In the presence
reaction mixture was 1 mL, including 4% ethanol. After the of the acetate anion, the reaction proceeded whereas essen-
reaction, 2-phenyl-2-propanol was added as an internal stan- tially no reaction was observed in the absence of acetic acid.
dard and the resulting reaction mixture was extracted with The conditions with 100 mM acetate anion gave the maximum
2 mL dichloromethane. The organic layer was washed with dis- turnover of 83 3 min−1. When the same reaction was carried
tilled water. After removal of the solvent under reduced out at a high concentration of sodium sulfate (up to 500 mM)
pressure at 30 °C, 50 μL dichloromethane was added to the to examine the effect of ionic strength, no reaction was
product. This solution was analysed by gas chromatography observed. The circular dichroism (CD) spectra of P450SPα in an
using a cyclosil-β column (Agilent Technologies, Inc.). The aqueous solution of sodium acetate (pH 7.0) at various acetate
absolute configuration of styrene oxide was determined by anion concentrations are shown in Fig. 3. The molar ellipticity
Fig. 2 Turnover rate for Russig’s blue formation catalysed by wild-type P450SPα (solid triangles connected with a solid line) and wild-type P450BSβ
(open circles connected with a solid line) in the presence of different concentrations of the acetate anion. Error bars represent the standard devi-
ation of at least five measurements. The reaction scheme on the right of the figure shows a plausible reaction mechanism for the formation of
Russig’s blue.
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[θ] at 222 nm, which is particularly sensitive to changes in the
α-helical structure of proteins, did not change even at a high
concentration of the acetate anion (Fig. 3 inset), suggesting
that the acetate anion did not induce structural changes in
P450SPα. These results show that the acetate anion serves as a
decoy molecule. We presume that the acetate anion interacts
with Arg-241 of P450SPα (Fig. 4), with its carboxylate serving as
a general acid–base catalyst for the generation of an active
species using hydrogen peroxide. P450BSβ also catalysed
1-methoxynaphthalene hydroxylation, and the maximum turn-
over rate reached 79 4 min−1 in the presence of 400 mM
acetate anion. Notably, the catalytic activity of P450SPα at low
acetate anion concentrations was higher than that of P450BSβ
,
Fig. 3 CD spectra of wild-type P450SPα in an aqueous solution of
sodium acetate. The pH was adjusted to 7.0 with hydrochloric acid.
whereas there was no remarkable difference in the maximum
turnover rate. A comparison of the active site of P450SPα with
that of P450BSβ shows that the former has a smaller active site
cavity, mainly due to the presence of Phe-288; the corres-
ponding amino acid in P450BSβ is Gly-290 (Fig. 5). According to
the crystal structure of the substrate-bound form of P450SPα
,
the edge of Phe-288 is close to the α-position of palmitic acid,
the carboxylate of which interacts with Arg-241 (Fig. 5A),
suggesting that the methyl group of the acetate anion may
interact with Phe-288.22 Based on the comparison between
amino acid residues in the active sites of P450SPα and P450BSβ
,
we decided to replace the Phe-288 residue of P450SPα with
glycine (F288G mutant). Compared with that of wild-type
P450SPα, the catalytic activity of the F288G mutant was lower at
a low concentration of the acetate anion (Fig. 6). We also pre-
pared a G290F mutant of P450BSβ and found that the catalytic
activity of this mutant increased especially at a low concen-
tration of the acetate anion. These results suggest that the Phe-
288 residue of P450SPα is a key residue in the reaction using
the acetate anion as a decoy molecule. The effect of using a
series of carboxylic acids on the hydroxylation of 1-methoxy-
Fig. 4 A plausible active site structure of P450SPα interacting with an
acetate anion.
Fig. 5 Active-site structure of the palmitic acid-bound form of P450SPα (A, PDB code: 3AWM) and P450BSβ (B, PDB code: 1IZO). The solvent accessi-
ble surface area is shown as a grey wire-frame.
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Fig. 6 Turnover rate for Russig’s blue formation catalysed by P450SPα WT (A, solid triangles connected with a solid line), P450SPα F288G mutant
(A, open triangles connected with a broken line), P450BSβ WT (B, open circles connected with a solid line) and P450BSβ G290F mutant (B, solid
circles connected with a broken line).
naphthalene is shown in Fig. 7. P450SPα and the G290F mutant P450SPα do not. This observed tendency also supports the con-
of P450BSβ tend to prefer short-alkyl-chain carboxylic acids as clusion that the Phe-288 residue in the active site of P450SPα is
decoy molecules, whereas P450BSβ and the F288G mutant of important for the binding of the acetate anion to the active
Fig. 7 Turnover rate for Russig’s blue formation catalysed by wild-type P450SPα (A), the F288G mutant of P450SPα (B), wild-type P450BSβ (C) and the
G290F mutant of P450BSβ (D) in the presence and the absence of 5 mM carboxylic acids; acetic acid (C2), propionic acid (C3), butanoic acid (C4),
pentanoic acid (C5), hexanoic acid (C6), heptanoic acid (C7) and octanoic acid (C8). The reaction without a carboxylic acid is also shown (None).
Error bars represent the standard deviation of at least three measurements.
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Table 1 Catalytic activity and selectivity for styrene oxidation by P450 in the presence of acetic acid
Concentration of the
acetate anion (mM)
Cytochrome P450
P450BSβWT
Turnover (102 min−1
)
ee (%)
Config.
SO : PAA
20
100
500
1000
20
100
500
1000
20
100
500
1000
20
100
500
1000
1.4 0.1
3.2 0.1
5.7 0.2
5.9 0.3
1.7 0.1
2.9 0.1
4.0 0.1
3.8 0.1
1.1 0.1
2.4 0.1
4.0 0.1
3.3 0.1
0.5 0.1
1.4 0.1
2.4 0.1
2.5 0.1
71
74
74
74
34
33
30
30
31
32
34
34
9
S
S
S
S
S
S
S
S
R
R
R
R
S
S
S
S
75 : 25
76 : 24
77 : 23
77 : 23
70 : 30
70 : 30
69 : 31
68 : 32
80 : 20
78 : 22
78 : 22
77 : 23
74 : 26
74 : 26
73 : 27
72 : 28
P450BSβG290F
P450SPαWT
P450SPαF288G
9
10
14
site. The maximum initial turnover rates of P450BSβ and the arginine residue located close to the heme and accelerates the
F288G mutant of P450SPα in the presence of a series of car- generation of the active species. A co-crystal structure analysis
boxylic acids were higher than those of P450SPα and the G290F of P450SPα and P450BSβ with an acetate anion would be
mutant of P450BSβ (Fig. 7), suggesting that access of non-native required to understand further the reaction at high acetate
substrates would be rather hampered by the Phe-288 residue anion concentrations.
in the active site of P450SPα. In this case, the smaller decoy
molecules would be more suitable from the viewpoint of
access of non-native substrates. This could also explain why
the short-alkyl-chain carboxylic acids are rather effective as
Conclusions
We have demonstrated that wild-type P450SPα and P450BSβ can
catalyse the oxidation of non-native substrates by performing
the reaction at a high concentration of the acetate anion. The
decoy molecules in the case of P450SPα and the G290F mutant
of P450BSβ. To expand our strategy of using the acetate anion
to other non-native substrates, epoxidation of styrene was
carried out. Although we have reported that P450SPα and
P450BSβ catalyse styrene epoxidation in the presence of car-
boxylic acids such as heptanoic acid and hexanoic acid,14,16
the reaction using the acetate anion as a decoy molecule had
not been studied. The epoxidation of styrene also proceeded in
the presence of a high concentration of the acetate anion
(Table 1). The catalytic activity was improved by increasing the
concentration of the acetate anion, whereas the product
selectivity (styrene oxide/phenylacetaldehyde) and stereo-
selectivity were not significantly affected by the acetate anion
concentration (Fig. S2†). It is important to note here that the
turnover rate of wild-type P450BSβ with 1 M acetate anion
reached 590 min−1, which is much higher than that of wild-
type P450BSβ with 20 mM hexanoic acid (330 min−1).14 Further-
more, to our knowledge, this is the highest initial turnover for
styrene oxidation among the cytochrome P450s using hydro-
gen peroxide reported so far.16,23,24 Although we could not esti-
mate the binding constants of the acetate anion toward
P450SPα or P450BSβ because their UV-Vis spectra did not
change upon increasing the concentration of the acetate
anion, we presume that the acetate anion interacts with the
Fig. 8 Genetic family tree (left) and ClustalW alignment of an amino
acid sequence (right) of the CYP152 family of enzymes.25 The sequence
number of CYP152A1 is shown on the top of the amino acid sequence.
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best initial turnover rate for styrene epoxidation catalysed by 10 O. Shoji and Y. Watanabe, J. Biol. Inorg. Chem., 2014, 19,
P450BSβ in the presence of 1 M acetate anion reached 590 529–539.
30 min−1. Although further studies are required, this strategy 11 D. S. Lee, A. Yamada, H. Sugimoto, I. Matsunaga,
is also expected to be applicable to most of the CYP152 family
of enzymes, because the arginine residue in the active site is
H. Ogura, K. Ichihara, S. Adachi, S. Y. Park and Y. Shiro,
J. Biol. Chem., 2003, 278, 9761–9767.
highly conserved among the 29 CYP152s reported thus far 12 K. Piontek, E. Strittmatter, R. Ullrich, G. Grobe,
(Fig. 8). We believe that our simple strategy of using the
acetate anion could be applicable to the oxidation of non-
M. J. Pecyna, M. Kluge, K. Scheibner, M. Hofrichter and
D. A. Plattner, J. Biol. Chem., 2013, 288, 34767–34776.
native substrates with the whole set of CYP152 enzymes and 13 E. Strittmatter, C. Liers, R. Ullrich, S. Wachter,
also with heme proteins having an arginine residue in their
active site.
M. Hofrichter, D. A. Plattner and K. Piontek, J. Biol. Chem.,
2013, 288, 4095–4102.
14 O. Shoji, T. Fujishiro, H. Nakajima, M. Kim, S. Nagano,
Y. Shiro and Y. Watanabe, Angew. Chem., Int. Ed., 2007, 46,
3656–3659.
15 T. Fujishiro, O. Shoji and Y. Watanabe, Tetrahedron Lett.,
2010, 52, 395–397.
Acknowledgements
This work was supported, in part, by Grants-in-Aid-for Scientific
Research (S) to Y. W. (24225004), for Young Scientists (A) to 16 T. Fujishiro, O. Shoji, N. Kawakami, T. Watanabe,
O. S. (26708018) and Grants-in-Aid for Scientific Research on
Innovative Areas “Molecular Activation Directed toward Straight-
H. Sugimoto, Y. Shiro and Y. Watanabe, Chem. – Asian J.,
2012, 7, 2286–2293.
forward Synthesis” to O. S. (25105724) from the Ministry of Edu- 17 O. Shoji, C. Wiese, T. Fujishiro, C. Shirataki, B. Wünsch
cation, Culture, Sports, Science and Technology (Japan).
and Y. Watanabe, J. Biol. Inorg. Chem., 2010, 15, 1109–1115.
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19 I. Matsunaga, A. Ueda, T. Sumimoto, K. Ichihara, M. Ayata
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