Dual-Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Article abstract of DOI:10.1002/anie.201801592

We report a unique strategy for the development of a H₂O₂-dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non-native substrates with the assistance of dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl fatty acyl)-l-amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid–base catalyst in the activation of H₂O₂. This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450–H₂O₂ system previously reported. This work provides the first example of the activation of the normally H₂O₂-inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

Full text of DOI:10.1002/anie.201801592

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Accepted Article
Title: The Use of Dual-Functional Small Molecules in Generating an
Efficient Cytochrome P450BM3 Peroxygenase
Authors: Nana Ma, Zhifeng Chen, Jie Chen, Jingfei Chen, Cong Wang,
Haifeng Zhou, Lishan Yao, Osami Shoji, Yoshihito Watanabe,
and Zhiqi Cong
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201801592
Angew. Chem. 10.1002/ange.201801592
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10.1002/anie.201801592
Angewandte Chemie International Edition
COMMUNICATION
The Use of Dual-Functional Small Molecules in Generating an
Efficient Cytochrome P450BM3 Peroxygenase
Nana Ma,† Zhifeng Chen,† Jie Chen,† Jingfei Chen, Cong Wang, Haifeng Zhou, Lishan Yao, Osami
Shoji, Yoshihito Watanabe, and Zhiqi Cong*
Abstract: We report a unique strategy for the development of a H2O2-
dependent cytochrome P450BM3 system, which catalyzes the
monooxygenation of non-native substrates with the assistance of
dual-functional small molecules (DFSMs), such as N-(ω-imidazolyl
peroxygenases, most native P450s lack the structural properties
required to activate H2O2.[4] Only a few native P450s, including
P450spα, P450BSβ, P450CLA, and P450 OleT, can efficiently utilize
H2O2 through a substrate-assisted reaction mechanism for the
hydroxylation or decarboxylation of fatty acids.[5] In view of the
significant potential of peroxygenases as practical biocatalysts,[6]
many efforts have been made to develop artificial H2O2-
dependent P450s. Several P450s, such as P450BM3 and
P450cam, have been engineered into their peroxygenase mode
through site-directed mutagenesis or directed evolution.[7] In
addition, the natural H2O2-dependent P450BSβ and P450spα have
been observed to oxidize non-native substrates in the presence
of decoy molecules.[8]
fatty acyl)-L-amino acids. The acyl amino acid group of DFSM is
responsible for bounding to enzyme as an anchoring group, while the
imidazolyl group plays the role of general acid-base catalyst in the
activation of H2O2. This system afforded the best peroxygenase
activity for the epoxidation of styrene, sulfoxidation of thioanisole, and
hydroxylation of ethylbenzene among those P450-H2O2 system
previously reported. This work provides the first example of the
activation of the normally H2O2-inert P450s through the introduction of
an exogenous small molecule. This approach improves the potential
use of P450s in organic synthesis as it avoids the expensive
consumption of the reduced nicotinamide cofactor NAD(P)H and its
dependent electron transport system. This introduces a promising
approach for exploiting enzyme activity and function based on direct
chemical intervention in the catalytic process.
Cytochrome P450s (CYP) are promising biocatalysts for
synthetic applications based on their versatile catalytic
monooxygenation of a variety of organic substrates, in reactions
such as hydroxylation, epoxidation, and sulfoxidation.[1] However,
most native P450s require reduced nicotinamide cofactor
NAD(P)H and a complex system of electron transport partners to
activate molecular oxygen, which limits their application in organic
synthesis.[1a,2] An alternative to these expensive cofactors is low-
cost H2O2, which can be used directly to exploit the catalytic
potential of the P450s. However, the peroxide shunt pathway is
generally inefficient in driving P450 catalysis in comparison with
those normal NADPH-dependent activity.[3] Unlike natural
Figure 1. A) The general accepted catalytic mechanism of AaeUPO
peroxygenase, in which Glu196 plays the role of general acid-base catalyst in
activating H2O2, resulting in compound I. B) Active site structure of P450BM3
bound to PFC9-L-Trp through multiple hydrogen-bonding interactions (PDB No.
[*]
N. Ma, J. Chen, Dr. J. Chen, Dr. C. Wang, Prof. Dr. L. Yao, Prof. Dr.
Z. Cong
CAS Key Laboratory of Biofuels, and Shandong Provincial Key
Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and
Bioprocess Technology, Chinese Academy of Sciences
Qingdao, Shandong, 266101(China)
3WSP). C) Active site structure of AaeUPO (PDB No. 2YOR). D) Schematic of
the P450BM3-H2O2 system, showing the use of DFSMs with an anchoring
group and an acid-base catalytic group in this study.
E-mail: congzq@qibebt.ac.cn
N. Ma, J. Chen
University of Chinese Academy of Sciences
Beijing, 100049 (China)
Z. Chen, Prof. Dr. H. Zhou
Hubei Key Laboratory of Natural Products Research and
Development, College of Biological and Pharmaceutical Sciences
China Three Gorges University
Yichang, Hubei, 443002 (China)
Dr. O. Shoji, Prof. Dr. H. Watanabe
The catalytic mechanism of natural peroxidases and
peroxygenases has been well discribed.[6a-b] For instance, the
peroxygenase from Agrocybe aegerita (AaeUPO) uses its distal
Glu196 to promote the deprotonation of the peroxocomplex and
the subsequent heterolytic cleavage of the O-O bond, resulting
in the active Compound I that is responsible for substrate
oxidation (Fig. 1A). It is generally accepted that the key histidine
or glutamic acid residues on the distal side of the heme serve as
general acid-base catalysts in the H2O2-activation step of
peroxygenases or peroxides.[9] In addition, although the crystal
structures of myoglobin and P450s reported to date haven’t
revealed such residues on the distal side of heme, Watanabe et
al. demonstrated that myoglobin and P450s can be modified
Department of Chemistry, Graduate School of Science, Nagoya
University
Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan)
These authors contributed equally to this work.
[†]
Supporting information for this article is available on the WWW
under http://www.angewandte.org.
This article is protected by copyright. All rights reserved.

10.1002/anie.201801592
Angewandte Chemie International Edition
COMMUNICATION
towards peroxidase or peroxygenase through the introduction of
an acid-base residue via site-directed mutagenesis.[10] However,
it is difficult to always ensure that this residue is directed to the
correct position for activity as a direct acid-base catalyst. We
respectively, were significantly faster than the 14 µmol·min-
1·(µmol P450)-1 for F87A alone over a 1-min reaction (Fig. S6-S7).
These results suggest that the DFSMs contributed to the
enzymatic epoxidation of styrene by H2O2. However, Im-C7-Phe
and Im-C8-Phe yieled unchanged and reduced TON, respectively
(Entries 4-5, Fig. S5). This is consistent with previous reports, in
which longer N-(ω-imidazolyl) fatty acids and their acyl amino acid
derivatives (C10 and C12) were shown to inhibit P450BM3.[15,19]
For a DFSM to perform the role of acid-base catalyst in the
activation of H2O2, we reasoned that the terminal imidazolyl group
has to be located at the suitable position in the active site. The
length of the DFSM may therefore be a critical factor. The above
results support this hypothesis.
recently resolved
perfluorononanoyl-
a
co-crystal of P450BM3 and N-
L
-tryptophan[11] (called a “decoy molecule”,
which efficiently reforms the active site of P450BM3 by partially
occupying the substrate tunnel[12]), in which the distance from the
terminus of the decoy molecule to the iron heme was 8.5 Å (Fig.
1B). Indeed, this distance is similar to those observed for H2O2-
dependent peroxides and peroxygenases, e.g., 5.9 Å between
Glu196 and the iron heme in AaeUPO4a (Fig. 1C), and 6.0 Å
between His42 and the iron heme in HRP[13]. Thus, we presumed
that if an acid-base group was inserted at the end of decoy
molecule, it may play the role of general acid-base catalyst in
promoting H2O2-activation of P450BM3. Herein, we report a
unique and efficient approach for generating peroxygenase
activity from P450BM3 using dual-functional small molecules
(DFSMs), which have a basic group at one end and an anchoring
group at the other end (Fig. 1D). The peroxygenase activity of the
H2O2-inert P450BM3 was drastically improved in the presence of
To examine the role of the imidazolyl group of the DFSMs,
we performed styrene epoxidations using various molecules
without a terminal acid-base functional group (Fig. 2 and S8).
These decoy molecules, either perfluorinated carboxylic acids
(PFC8 - PFC10) or their phenylalanine derivatives (PFC8-
L-Phe -
PFC10- -Phe), did not yield positive results. The short-chain fatty
L
acids (C8 - C10) produced a minor improvement in the TON to
~2-fold level,[20] however, the corresponding phenylalanine
derivative (C9-L-Phe) led to the reduction of the TON by 50%. We
also prepared ω-pyrrolyl acyl phenylalanine (Pyr-C6-Phe) using
pyrrolyl instead of imidazolyl. This structural analogue of Im-C6-
Phe could not increase the peroxygenase activity of F87A. These
results strongly suggest that the imidazolyl group plays a key role
in the H2O2-dependent epoxidation of P450BM3.
the DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine (Im-C6-
Phe). The maximum turnover numbers reached 2733, 4910, and
730 for the epoxidation of styrene, sulfoxidation of thioanisole,
and hydroxylation of ethylbenzene.
Dual-Functional Small Molecules (DFSMs)
The anchoring group of the DFSM is also crucial for the
epoxidation of styrene in this system. Molecules without an
anchoring group (Fig. 2), e.g., 1-methylimidazole (1-Me-Im) and
1-hexylimidazole (1-Hex-Im), were demonstrated to inhibit the
epoxidation of styrene (Fig. S8F). It is likely that the free imidazolyl
simply coordinates with the iron heme without the pulling effect of
the anchoring group. All reactions by the DFSMs with amino acid
groups yielded higher TONs and initial rates than that by F87A
alone (Entries 6-12, Fig. S9). According to previous reports, this
may be due to the DFSMs containing an acyl amino acid group,
which has a higher binding affinity for P450BM3.[11,21] It is
therefore favorable to target the DFSMs towards the active center,
as this is a relatively stable position from which it can better
perform its catalytic role.
R
O
O
N
N
N
OH
N
N
H
OH
n
n
O
Im-C5 ~ ImC8: n = 0, 1, 2
Im-C5-amino acid ~ Im-C8-amino acids: n = 0, 1, 2, 3
R:
Met
Ala
Phe
Ile
Leu
Val
Trp
S
CH3
NH
Without anchoring group
Without terminal acid-base group
Ph
O
O
O
N
N
N
N
OH
OH
n
O
1-Hex-Im
C9-Phe
1-Me-Im
N
C8 ~C10: n = 0, 1, 2
O
Ph
OH
Ph
O
F
F
O
F
F F F F F
F
F F F F F
O
F
F
F
F
OH
OH
n
N
H
n
N
H
F
F F F F F F F
F
F F
F F F F F
O
Pyr-C6-Phe
PFC8 ~ PFC10: n = 0, 1, 2
PFC8-Phe ~ PFC10-Phe: n = 0, 1, 2
The DFSM-facilitated P450BM3-H2O2 system was then
applied to the wild-type enzyme and its variants including F87G,
F87V, F87I, and F87L (Entries 13-22, Fig. S10). In the absence
of the DFSMs, F87V showed the best catalytic TON (120) for
styrene epoxidation. F87G and F87I produced similar catalytic
TONs, while F87L and the wild-type showed the worst catalytic
TONs. Upon addition of Im-C6-Phe, the catalytic TON increased
2 - 3-fold for all the enzymes except F87G and F87A, which
exhibited more than 20-fold improvement. These results
confirmed a previous report that the residue size at position 87
plays a critical role in the ability of P450BM3 to use H2O2.[18a] This
suggests that when researchers design a DFSM-driven P450-
H2O2 system, they should pay attention to the significant effect of
free space near the iron heme on the activity of the system.
The above results validated our hypothesis that the DFSMs
could improve the catalytic activities of the P450-H2O2 system. To
further prove that the peroxygenase activity is derived from the
presence of the DFSMs, we prepared a H2O2-inactive P450BM3
mutant through the substitution of Thr268. The distal residue
Thr268 has been implicated in various roles related to the active
Figure 2. Structures of the dual-functional small molecules and mono-functional
small molecules (as controls) used in this study.
We first prepared N-(ω-imidazolyl) fatty acids (Im-Cn: n= 5-
8) and their amino acid derivatives according to the previous
reports (Fig. 2).[15] The epoxidation of styrene catalyzed by the
F87A mutant of the heme domain of P450BM3 was chosen as the
reaction model system (Fig. S1-S2). The formation of styrene
oxide product was estimated with a well-established colorimetric
method (Fig. S3).[16] The F87A mutant exhibited a catalytic
turnover number (TON) of 42 in 20 mM H2O2[17-18] at 25°C in the
absence of DFSM (Table 1). The TON was slightly increased by
~1.3-fold on average upon addition of Im-Cn (Fig. S4). However,
the TON was significantly improved to 220 when using Im-C5-Phe,
with a TON of up to 1377 observed with Im-C6-Phe over a 30-min
reaction (Entries 2-3, Fig. S5), which is ~30-fold higher than that
of F87A alone. The product formation rates (PFR) of 32 and 796
µmol·min-1·(µmol P450)-1 for Im-C5-Phe and Im-C6-Phe,
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10.1002/anie.201801592
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COMMUNICATION
oxygen species in the catalytic cycle of P450BM3, including
proton donation, dioxygen activation, and stabilization of the oxy-
ferrous or hydroperoxyl catalytic intermediates.[1e] We envisaged
that the use of valine to replace the Thr268 of F87A would
eliminate its ability to utilize H2O2. Indeed, no oxidized products
were detected in reactions containing styrene and F87A-T268V in
20 mM H2O2 at 25°C. However, upon addition of Im-C6-Phe,
styrene oxide was produced at an initial PFR of 125 µmol·min-
1·(µmol P450)-1 in a 1-min reaction, with a total TON of 382 for a
30-min reaction (Entries 23-24, Fig. S11). This result strongly
supported the hypothesis that DFSM directly participates in the
activation of H2O2, which in turn facilitates the peroxygenase
activity of P450BM3.
P450BM3 and its variants F87L, F87I, and F87V yielded (S)-(+)-
styrene oxide with an enantiomeric excess ranging from 1-67%
as the major product in 20 mM H2O2, while F87A and F87G
yielded (R)-(+)-styrene oxide with an enantiomeric excess of 7%
and 52%, respectively (Table 1, Fig. S12). These results are
similar to those observed for the epoxidation of 3-chlorostyrene,
in which the enantioselectivity is mainly determined by the residue
size at position 87.[22] Upon addition of Im-C6-Phe, the ratio of (R)-
(+)-styrene oxide increased in most cases except for the wild-type
and F87L enzymes, which exhibited the reverse trend. The
combination of F87A and Im-C6-Phe achieved the best
enantiomeric excess of 84% among the F87 mutants. The
enantiomeric excess of the (R)-enantiomer was further improved
to 91% for the combination of F87A-T268V/Im-C6-Phe. To our
knowledge, this represents the highest enantioselectivity of
styrene epoxidation achieved by P450s.[7-9,10b,23] In addition,
DFSMs with various acyl amino acid groups produced different
effects on the change in enantioselectivity (Table 1, Fig. S13).
Table 1. H2O2-dependent epoxidation of styrene catalysed by P450BM3 in
the absence/presence of DFSMs[a,b]
Entry
1
Enzyme/DFSM
F87A/-
PFR[c]
14±1
32±1
796±14
-
TON[d]
42±2
ee[e]
7
O
F87A
Im-C6-Phe
2
F87A/Im-C5-Phe
F87A/Im-C6-Phe
F87A/Im-C7-Phe
F87A/Im-C8-Phe
F87A/Im-C6-Gly
F87A/Im-C6-Ala
F87A/Im-C6-Val
F87A/Im-C6-Met
F87A/Im-C6-Leu
F87A/Im-C6-Trp
F87A/Im-C6-Ile
F87G/-
220±9
1377±27
42±2
-
S
S

H2O2
3
84
-
TON: 3436; ee: 32% (S)
4
OH
F87A-T268V
Im-C6-Phe
OH
OH
5
-
4±2
-

+
+
H2O2
6
15±1
30±1
43±1
154±12
358±14
137±11
628±7
26±1
275±8
23±2
99±2
22±2
96±2
4±1
60±1
20
27
21
66
68
46
77
52
73
-67
-62
-66
-40
-14
-22
<-1
-5
TON: 319; ee: 14% (R)
TON: 40
TON: 14
7
90±2
8
136±6
382±12
596±2
726±14
1004±30
53±2
Scheme 1. Sulfoxidation and hydroxylation reactions catalyzed by P450BM3
mutants in the presence of Im-C6-Phe.
9
10f
11f
12
13
14
15
16
17
18
19
20
21
22
23
24
The DFSM-facilitated P450BM3 peroxygenase system also
worked well for the sulfoxidation of thioether and the hydroxylation
of ethylbenzene (Scheme 1). The oxidation of thioanisole
catalyzed by F87A/Im-C6-Phe yielded sulfoxide as the sole
product according to GC-MS analysis (Fig. S14-S17). The PFR
was 888 µmol·min-1·(µmol P450)-1 for the 1-min reaction, and the
catalytic TON reached 3436 for the 30-min reaction, resulting in
an almost 35-fold improvement in the TON over F87A alone. The
enantiomeric excess was 32% for (S)-(+)-sulfoxide in the
presence of Im-C6-Phe. The hydroxylation of ethylbenzene
yielded 1-phenylethanol as the major product accompanying
small amounts of 2-phenylethanol and 4-ethylphenol (Fig. S18-
S21). The total catalytic TON of the F87A-T268V/Im-C6-Phe
combination was drastically improved to 373, while no oxidative
product was detected for F87A-T268V alone. The PFR of 1-
phenylethanol was estimated at 80 µmol·min-1·(µmol P450)-1 for
the 1-min reaction, and the enantiomeric excess was 14% for (R)-
(+)-1-phenylethanol.
F87G/Im-C6-Phe
F87V/-
1190±26
120±4
250±6
52±2
F87V/Im-C6-Phe
F87I/-
F87I/Im-C6-Phe
F87L/-
176±8
15±1
F87L/Im-C6-Phe
WT/-
27±2
10±1
9±1
30±2
13±2
WT/Im-C6-Phe
AV[g]/-
AV[g]/Im-C6-Phe
44±3
nd[h]
nd[h]
-
125±4
382±2
91
The observed fast PFRs and the relative lower TONs for
these reactions indicated that enzyme stability still remains a
major challenge for the P450 peroxygenase system.[24] Actually,
the F87A heme decayed within 1 h in 20 mM H2O2 in a pseudo
first order reaction with a rate of 4.74 × 10-4 s-1. Upon addition of
Im-C6-Phe, the degradation rate increased one order of
magnitude up to 5.08 × 10-3 s-1. It reduced to 1.86 × 10-3 s-1 after
the addition of styrene (Fig. S22). These results suggested that
DFSMs promote the activation of H2O2 to form active species,
most of which are used for the final epoxidation reaction but a
considerable portion are still consumed through enzyme
[a] Reaction conditions: P450BM3 (0.5 µM), Styrene (4 mM), H2O2 (20 mM),
DFSM (500 µM), in pH 8.0 phosphate buffer. [b]Phenyl acetaldehyde was
observed by GC-MS as a minor reaction product (5% molar ratio) to styrene
oxide (Fig. S7). [c]PFR: Product formation rates were estimated for 1-min
reactions as µmol·min-1·(µmol P450)-1. [d]TON: Turnover numbers were
estimated for 30-min reactions. [e]ee: the enantiomeric excess (%) yield of
(R)-(+)-styrene oxidedetermined by chiral HPLC. [f]For these reactions, Im-
C6-Leu and Im-C6-Trp were used at 5 mM rather than 500 µM. [g] AV: F87A-
T268V. [h] nd: not detected.
It is important to note that the DFSMs significantly influenced
the enantioselectivity of styrene epoxidation. The wild-type
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10.1002/anie.201801592
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COMMUNICATION
119; (d) M. A. Rude, T. S. Baron, S. Brubaker, M. Alibhai, S. B. Del
Cardayre, A. Schirmer, Appl. Environ. Microbiol. 2011, 77, 1718-1727.
(a) Y. Wang, D. Lan, R. Durrani, F. Hollmann, Curr. Opin. Chem. Biol.
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degradation. Anyhow, the problem of enzyme stability can be
solved through protein engineering[25] or the use of in-situ H2O2
formation to control the H2O2 concentration[26]. We also observed
that the TONs increased to 2733, 4910, and 730 for the lengthier
reactions of styrene epoxidation, thioanisole sulfoxidation, and
ethylbenzene hydroxylation at 4 °C. This demonstrated that the
catalytic efficiency of the system could be further improved by
enhancing the stability of the enzyme.
[6]
[7] (a) P. C. Cirino, F. H. Arnold, Angew. Chem. Int. Ed. 2003, 42, 3299-3301;
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104, 1185-1194.
In summary, we have constructed a unique approach for
generating P450BM3 peroxygenase activity through the use of
exogenous DFSMs. Our system achieved the best peroxygenase
activity for the epoxidation of styrene, sulfoxidation of thioanisole,
and hydroxylation of ethylbenzene among those P450-H2O2
system reported to date.[7-9,10b,14] We believe that this system can
be further improved by optimizing the structure of the DFSMs in
combination with rational design of the enzyme. Considering that
quite a few of the cytochrome P450s have a large and vacant
substrate pocket, such optimization would offer further avenues
for the development of various P450 peroxygenase systems. The
present study emphasizes how the modification of an enzyme’s
activity and function through direct chemical intervention can
influence its catalytic process. Further investigations of the
detailed activation mechanisms involved in this system are now
in progress in our laboratory.
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Keywords: biocatalysis • peroxygenase • cytochrome P450s •
protein engineerings • oxidoreductases
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This article is protected by copyright. All rights reserved.

10.1002/anie.201801592
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Nana Ma, Zhifeng Chen, Jie Chen,
Jingfei Chen, Cong Wang, Haifeng
Zhou, Lishan Yao, Osami Shoji,
Yoshihito Watanabe, and Zhiqi Cong*
((Insert TOC Graphic here))
Page No. – Page No.
The Use of Dual-Functional Small
Molecules in Generating an Efficient
Cytochrome P450BM3 Peroxygenase
A dual-functional small molecules-based approach for efficiently generating a H2O2-
dependent cytochrome P450BM3 system to afford the best peroxygenase activity
for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of
ethylbenzene among the reported P450-H2O2 system to data.
This article is protected by copyright. All rights reserved.