Enhanced Activity and Substrate Specificity by Site-Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole

Article abstract of DOI:10.1002/open.201900157

P450 119 peroxygenase was found to catalyze the sulfoxidation of thioanisole and the sulfonation of sulfoxide in the presence of tert-butyl hydroperoxide (TBHP) for the first time with turnover rates of 1549 min^?1 and 196 min^?1 respectively. Several mutants were designed to improve the peroxygenation activity and thioanisole specificity by site-directed mutagenesis. The F153G/T213G mutant gave an increase of sulfoxide yield and a decrease of sulfone yield. Moreover the S148P/I161T/K199E/T214V mutant and the K199E mutant with acidic Glu residue contributed to improving the product ratio of sulfoxide to sulfone. Addition of short-alkyl-chain organic acids to the P450 119 peroxygenase-catalyzed sulfur oxidation of thioanisole was investigated. Octanoic acid was found to induce a preferred sulfoxidation of thioanisole catalyzed by the F153G/T213G mutant to give approximately 2.4-fold increase in turnover rate with a k_cat value of 3687 min^?1 relative to that of the wild-type, and by the F153G mutant to give the R-sulfoxide up to 30 % ee. The experimental control and the proposed mechanism for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in the presence of octanoic acid suggested that octanoic acid could partially occupy the substrate pocket; meanwhile the F153G mutation could enhance the substrate specificity, which could lead to efficiently regulate the spatial orientation of thioanisole and facilitate the formation of Compound I. This is the most effective catalytic system for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole.

Full text of DOI:10.1002/open.201900157

DOI: 10.1002/open.201900157
Full Papers
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Enhanced Activity and Substrate Specificity by Site-
Directed Mutagenesis for the P450 119 Peroxygenase
Catalyzed Sulfoxidation of Thioanisole
Xiaoyao Wei+,[a] Chun Zhang+,[a] Xiaowei Gao+,[a] Yanping Gao,[a] Ya Yang,[a] Kai Guo,[a] Xi Du,[a]
Lin Pu,*[b] and Qin Wang*[a]
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P450 119 peroxygenase was found to catalyze the sulfoxidation
of thioanisole and the sulfonation of sulfoxide in the presence
of tert-butyl hydroperoxide (TBHP) for the first time with
turnover rates of 1549 minÀ 1 and 196 minÀ 1 respectively. Several
mutants were designed to improve the peroxygenation activity
and thioanisole specificity by site-directed mutagenesis. The
F153G/T213G mutant gave an increase of sulfoxide yield and a
decrease of sulfone yield. Moreover the S148P/I161T/K199E/
T214V mutant and the K199E mutant with acidic Glu residue
contributed to improving the product ratio of sulfoxide to
sulfone. Addition of short-alkyl-chain organic acids to the P450
119 peroxygenase-catalyzed sulfur oxidation of thioanisole was
investigated. Octanoic acid was found to induce a preferred
sulfoxidation of thioanisole catalyzed by the F153G/T213G
mutant to give approximately 2.4-fold increase in turnover rate
with a kcat value of 3687 minÀ 1 relative to that of the wild-type,
and by the F153G mutant to give the R-sulfoxide up to 30% ee.
The experimental control and the proposed mechanism for the
P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in
the presence of octanoic acid suggested that octanoic acid
could partially occupy the substrate pocket; meanwhile the
F153G mutation could enhance the substrate specificity, which
could lead to efficiently regulate the spatial orientation of
thioanisole and facilitate the formation of Compound I. This is
the most effective catalytic system for the P450 119 peroxyge-
nase-catalyzed sulfoxidation of thioanisole.
1. Introduction
cofactors and complicated electron-transfer systems such as
NADH or NADPH in synthetic chemistry. In contrast to P450
monooxygenases, P450 peroxygenases employ hydroperoxide
or other peroxides as surrogate oxygen donors to catalyze the
oxygenation of substrates through a so-called shunt pathway,
and in principle make reaction much simpler in the absence of
NAD(P)H and redox protein partners.[4–5] However, excess
peroxides may deactivate P450s by heme destruction and
oxidative modification of amino residues, which makes the use
of the shunt pathway not productive. Furthermore, formation
of Compound I by the peroxide shunt pathway is restricted by
the size and shape of the substrate-binding pocket of the
P450s, which allows other competitive radical species to
generate secondary substrate oxidation.
Cytochrome P450 enzymes (P450s or CYPs) are a family of
hemoproteins that catalyze a wide range of oxidative reactions
including aliphatic and aromatic hydroxylation, olefin epoxida-
tion and sulfur oxidation in a regio- and stereoselective
manner.[1–3] This makes P450s highly promising candidates for
the production of fine chemicals that are difficult to synthesize
by standard chemical means. For many oxidative reactions,
P450s typically catalyze the insertion of one atom of molecular
oxygen (monooxygenation) into exogenous and endogenous
substrates through
a
classical two-electron oxotransfer
pathway.[3] The mechanism of P450 monooxygenases involves a
putative Compound I species, which is a known intermediate
formed in oxidative reactions of other heme-containing
enzymes.[1] One of the limitations to apply P450 monooxyge-
nases to biocatalysts is that the process requires expensive
P450 119 cloning from archaebacteria Sulfolobus acid-
ocaldarius contains the typical P450-fold with a relatively
compact structure.[6–7] The available crystal structure indicated
that CYP119 undergoes a significant conformational change in
the F/G region upon binding of medium substrates, which is
helpful to define the determinants of substrate specificity for
the subsequent modification of the protein to the desired
properties.[7] It was suggested that the extensive aromatic
cluster and entropic contribution could lead to the enhanced
[a] X. Wei,+ Dr. C. Zhang,+ Dr. X. Gao,+ Y. Gao, Y. Yang, Dr. K. Guo, X. Du,
Prof. Dr. Q. Wang
Department of Medicinal Chemistry, School of Pharmacy
Southwest Medical University
Luzhou, Sichuan, 646000, P. R. China
E-mail: wq_ring@hotmail.com
[b] L. Pu
[8]
thermal stability of CYP119. It is an attractive system for the
formation of the Compound I species by the shunt pathway in
mechanistic investigations of the P450 catalytic cycle.[9] Gen-
erally, the thermal stability of CYP119 does not depend on the
iron spin state or the active site of the substrate-binding
pocket.[6] Though both the native electron donor partners and
the endogenous substrate for CYP119 are still unknown, its site-
Department of Chemistry
University of Virginia
Charlottesville, VA 22904–4319, USA
E-mail: lp6n@virginia.edu
[+] These authors contributed equally to this work
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900157
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[20]
directed mutant can hydroxylate lauric acid by putidaredoxin
(Pd) and putidaredoxin reductase (PdR) with NADH as surrogate
redox partners.[10] The T214 V/D77R mutant with a kcat value of
8.8 minÀ 1 showed 15-fold enhanced activity over the wild-type.
4-one by directed evolution.
There are only few cases
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reported for the sulfoxidation of thioether by using P450
peroxygenase. For example, P450BSβ peroxygenase isolated from
Bacillus subtilis was reported with an enhanced turnover rate for
the sulfoxidation of thioanisole in the presence of carboxylic
acids by utilizing hydrogen peroxide.[21] The P450BM3 F87 A
mutant peroxygenase also worked well for the sulfoxidation of
thioanisole by using dual-functional small molecules in the
presence of H2O2.[22] To our knowledge, no sulfoxidation of
thioanisole has been achieved by using P450 119 peroxyge-
nase.
In the present work, we report that for the first time the
thermophilic P450 119 peroxygenase can catalyze the sulfur
oxidation of thioanisole in the presence of TBHP. Site-directed
mutations of CYP119 and screening assays of the reaction
conditions have been conducted to enhance the peroxygena-
tion activity and substrate specificity for the sulfoxidation of
thioanisole.
[10]
It was suggested that the T214 V mutation should increase
the rate of hydroxylation by modifying the size and shape for
the substrate binding, whereas the D77R mutation should
improve the affinity between CYP119 and the redox protein
partner. CYP119 was initially isolated from S. solfataricus, which
is closely related to S. acidocaldarius, so the electron transfer
proteins or proliferating cell nuclear antigen from this thermo-
philic organism were reconstituted into P450 119 monooxyge-
nation, in which the homologous recombination from sulfur-
containing microorganism makes hydroxylation of lauric acid
work.[11–13]
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P450 119 was also found to catalyze the peroxygenation of
styrene and substituted styrenes in the presence of H2O2 and
other peroxides. Styrene was peroxygenated to the correspond-
°
ing epoxides by using the wild-type P450 119 at 30 C and low
catalytic activity was observed with a rate of 0.6 minÀ 1, while
the P450 119 T214 V mutant was found to increase the H2O2- 2. Results and Discussion
dependent epoxidation rate by about 3-fold under the same
reaction conditions.[6] Niemeyer et al reported that P450 119
catalyzed the epoxidation of styrene with an increased turnover
rate of 78 minÀ 1 by using the optimized reaction conditions at
We first examined the sulfur oxidation of thioanisole 1 catalyzed
°
by the wild-type P450 119 peroxygenase at 35 C and pH 7.5 in
the presence of TBHP according to our previously optimized
reaction conditions.[16] Trace products were found in 10 min
under these conditions (Table 1, Entry 1). Since TBHP was
°
70 C and pH 8.5 in the presence of tert-butyl hydroperoxide
(TBHP)[13]. We reported that introduction of the T213G mutation
into the wild-type P450 119 enhanced the turnover rate for the
epoxidation of styrene with kcat value of 51.2 minÀ 1 and
Table 1. The results of the sulfur oxidation of thioanisole for P450 119 at
346.2 minÀ 1 respectively at 35 C and 70 C in the presence of
TBHP at pH 7.5.[14] The increased turnover rate might be mainly
due to the reduced steric hindrance of the T213G mutation,
which could improve the access of styrene and TBHP to the
catalytic site. The T213 M mutant can improve the enantiose-
lectivity for the epoxidation of cis-β-methylstyrenes, but it gave
°
°
°
35 C and pH 7.5 in presence of various concentrations of TBHP.
Entry
Conc. of TBHP
Yield (%)
Yield (%)
Conf.
2[a]
3[a]
2[b]
1[c]
2
3
4
5
2.5 mM
2.5 mM
8.0 mM
15.0 mM
20.0 mM
30.0 mM
trace
36.0
46.2
48.0
59.3
54.8
–
0.3
26.0
31.3
30.3
23.8
–
R
R
R
R
R
low turnover number with a rate of 32.9 minÀ 1 at 35 C under
°
the same reaction condition.[15] Our recent study showed that
the quadruple mutant S148P/I161T/K199E/T214 V (No.3-39)
obtained by using the directed evolution can significantly
improve the turnover number for the epoxidation of styrene
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[a] yield of 2 and 3 determined by GC-MS. [b] enantioselectivity
determined by HPLC and configuration determined by comparing the
HPLC data the literature 21. [c] reaction conditions from reference 16.
and its derivatives with up to 308.8 minÀ 1 at 35 C and pH 7.5 in
°
the presence of TBHP.[16] The proposed mechanism suggested
that the improved access of the substrate channel and the
increased affinity of the acid-base controlling in the catalytic
pocket of P450 119 peroxygenase might result in high efficiency
reported to increase the stability of P450 119 under the
peroxidation conditions,[13] we prolonged the incubation time.
Within 60 min, sufficient amounts of the products were
observed in this reaction. An incomplete conversion of 1 with
formation of the sulfoxide 2 (36% yield) was determined by GC-
MS analyses (Scheme 1 and Table 1, Entry 2), with the aid of an
internal standard calibration curve prepared by using acetophe-
none (Figure S1 in Surpporting Information). Since a high
concentration of TBHP relative to the concentrations of the
substrate and P450 119 can also be attributed to the improved
turnover rate of a peroxidation in an earlier activity assay, [13] we
investigated the sulfoxidation of 1 catalyzed by P450 119 with
various concentrations of TBHP. As shown in entries 3–6 of
Table 1, the yield of the sulfoxide 2 increased from 46% to 59%
for the formation of Compound
peroxygenation.
I and the subsequent
Chiral sulfoxides are important building blocks in medicinal
chemistry and pharmaceutical industry. Among several sulfur
oxidations catalyzed by cytochrome P450 enzymes,[17–18] an
auxiliary flavin containing cofactor and NADH-NADPH depend-
ent flavoenzymes need to be considered. For example, P450
monooxygenase CYP116B4 from Labrenzia aggregata exhibits
sulfoxidation activity towards alkyl-aryl sulfides with a distinc-
[19]
tive CYP-redox system.
The extensively studied P450BM3
naturally fused with an FAD/FMN redox partner was also
mutated for application in the sulfoxidation of 1-thiochroman-
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the basis of our previously reported mutants.[14–15] Earlier, we
found that introduction of glycine and methionine into the
Thr213 residue of the wild-type P450 119, giving the T213G and
T213 M mutants respectively, can enhance the turnover rate
and enantioselectivity for the peroxide-dependent styrene
epoxidation.[14] A histidine residue as a proximal or distal ligand
1
2
3
4
5
6
7
Scheme 1. Cytochrome P450 119-catalyzed sulfur oxidation of thioanisole in
the presence of TBHP.
plays
a vital role in high peroxidase and peroxygenase
activity.[5,23] We speculate that the site-directed mutation of
Thr213 to histidine as a distal side of P450 119 might enhance
P450 peroxygenase activity, in which the distal His213 plays
general acid-base function while the proximal Cys317 is still
maintained to ligate heme. In order to enhance the substrate
specificity of P450 119 for the sulfur oxidation of thioanisole,
molecular dynamics (MD) simulations were carried out and the
root-mean-square fluctuation (RMSF) of the whole protein and
residues was analyzed (Figure S3 in SI). We found that the F/G
loop from 148 to 163 residues constitute the significant binding
region to recognize substrate with more conformational
flexibility (Figure S3 in SI), while the Phe153 residue with phenyl
side chain is located into the region with large steric hindrance
and rigidity. We propose that the site-directed mutation of the
Phe153 residue in P450 119 to a small and flexible residue such
as glycine, alanine or valine might contribute to the special
recognition of thioanisole.
Molecular docking was used to investigate the sulfoxidation
of 1 with the wild-type P450 119 and the F153G/T213G mutant.
We modeled the docking of the thioanisole into the wild-type
P450 119 and the F153G/T213G mutant respectively. As shown
in Figure 1, the distances between the sulfur atom and the iron-
oxo species of the heme are 2.4 Å and 1.9 Å respectively
(Figure 1a and 1b), which suggests that the shorter the distance
of the sulfur atom from Compound I, the higher the peroxyge-
nation activity of the P450s for the sulfoxidation of 1. It should
be noted that when F153 was mutated into G153, the F/G helix
of the F153G residue became shorter, and the region near the
substrate center formed a loop structure to squeeze the
substrate closer to the active center (Figure 1b).
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with the increase of the TBHP concentration. Meanwhile
formation of the sulfone 3 was also observed in 23–31% yield,
and the ratio of the sulfoxide product to the sulfone did not
show significant change under these conditions. Complete
conversion of 1 by using P450 119 gave maximum yields of the
sulfoxide 2 and the sulfone 3 in 61% and 33% respectively in
the presence of 20 mM TBHP (Entry 4). An enantioselectivity of
16.9%ee was also observed for the sulfoxidation of 1, and the
configuration of the sulfoxide 2 was determined to be R by
HPLC (Daicel Chiralpak ODÀ H column) in comparison with the
data reported in the literature.[21] Neither 2 nor 3 was observed
without P450 119 or TBHP in the control assay for the sulfur
oxidation of 1.
The sulfoxidation of 1 by using P450 119 gave only trace
amount of the sulfone 3 (0.3% yield) in 2.5 mM TBHP (Table 1,
Entry 2). The initial rate for the formation of the sulfoxide 2 was
investigated in the oxidation of 1 catalyzed by P450 119 in the
presence of 20 mM TBHP. The kinetic constant for the
sulfoxidation of thioanisole was calculated from the resulting
regression equation. The wild-type P450 119 converted 1 into
the sulfoxide 2 with a kcat value of 1549 minÀ 1 and a Km value of
4.3 mM on the basis of the double-reciprocal plot (Figure S2a in
SI). The sulfoxide 2 was also used as the substrate for the
oxidation catalyzed by P450 119, and formation of the sulfone 3
was observed under the same reaction conditions (Scheme 2).
The kinetic constants kcat and Km for the oxidation of the
sulfoxide 2 catalyzed by P450 119 were 196 minÀ 1 and 6.3 mM
(see Figure S2b in SI). So, there were 8 times difference in the
turnover rate for the sulfoxidation of thioanisole and the
sulfonation of the sulfoxide. This is the first time that P450 119
was found to catalyze the sulfur oxidation of thioanisole in the
presence of TBHP, in which P450 119 maintained its peroxyge-
nation activity with the formation of a sulfoxide 2 and a sulfone
3, and moreover P450 119 showed its substrate specificity
between 1 and 2 with a different yield of 2 and 3.
Recently, we also reported that the quadruple mutant
S148P/I161T/K199E/T214V (No.3-39) obtained by using the
directed evolution can significantly improve the turnover
number for the peroxide-dependent epoxidation.[16] The model-
ling structures revealed that introduction of the acidic Glu
residue into the basic Lys199 residue located within the I helix
of the quadruple mutant could lead to the reconstruction of the
whole catalytic pocket,[16] which might be crucial for both the
peroxygenation activity and the substrate specificity. Therefore,
In order to improve the peroxygenation activity and
substrate specificity of P450 119 for the sulfur oxidation of
thioanisole, we designed several new site-directed mutants on
Scheme 2. Cytochrome P450 119-catalyzed the oxidation of the sulfoxide in
the presence of TBHP.
Figure 1. The molecular docking of the thioanisole 1 into (a) the wild-type
P450 119 and (b) the F153G/T213G mutant.
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we decided to examine whether a single K199E mutant could
influence the sulfoxidation of thioanisole in comparison with
the quadruple mutant. Similar to the construction, expression,
and purification of the above-mentioned mutants, the K199E
mutant was obtained with a typical P450s character in the UV/
Vis absorption spectrum (Figure S5 in SI). The sulfoxidation of 1
catalyzed by the No.3-39 mutant and the K199E mutant was
investigated. As shown in Table 2 (Entries 9 and 10), the
and 3 in 53.6% and 28.9% respectively (Entry 3) similar to the
results of the sulfur oxidation of 1 catalyzed by the wild-type
without carboxylic acid (Entry 1). A great difference in the 2/3
yield ratio was obtained with 79.1% versus 15.4% in the
presence of octanoic acid, indicating significant enhancement
in the peroxygenation activity and the substrate specificity
(Entry 4). Carboxylic acids with a phenyl group gave smaller
differences in the yields of the 2 and 3 with 47.3–47.8% and
37.9–40.9% respectively for the sulfur oxidation of 1 catalyzed
by P450 119 (Entries 5–6). These carboxylic acids examined
gave the R-sulfoxide 2 with increased enantioselectivity ranging
from 21.4% to 28.7% ee. Thus, P450 119 peroxygenases in the
sulfoxidation of 1 exhibit significant dependence on the alkyl-
chain length of the carboxylic acid additives.
We studied the octanoic acid-attached P450 119 system
with an adjusted pH value of 6.5 in potassium phosphate buffer
(50 mM). The optimized conditions were applied to the sulfur
oxidation of 1 catalyzed by the P450 119 mutants at pH 6.5 in
presence of TBHP and octanoic acid, and the results are
summarized in Table 4. As shown in Table 4, octanoic acid had
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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Table 2. The results of the sulfur oxidation of thioanisole for P450 119 and
°
its mutants at 35 C and pH 7.5 in presence of 20 mM TBHP.
Entry
Enzyme
Yield (%)
ee (%)
Conf.
2/3[a]
2[b]
2[c]
1
2
3
4
5
6
7
8
Wild-Type
T213G
T213M
T213H
F153G
F153A
F153V
F153G/T213G
No. 3–39
K199E
59.3/30.3
72.0/14.5
10.3/–
16.9
0.2
–
R
R
–
–
R
R
R
R
R
R
15.4/–
–
71.3/22.2
53.4/29.5
60.6/21.9
75.1/11.3
76.2/6.9
70.2/18.5
22.9
19.9
17.5
6.8
14.8
12.3
9
10
Table 4. The results of the sulfur oxidation of thioanisole for P450 119 and
its mutants at pH 6.5 with octanoic acid and pH 8.5 without octanoic acid.
sulfoxidation of 1 catalyzed by the No.3-39 mutant gave 76.2%
yield of the 2 and 6.9% yield of the 3, and the K199E mutant
retained similar yields of 2 and 3 with 70.2% and 18.5%
respectively. Thus, an acidic surrounding of the catalytic pocket
in the quadruple mutant and the K199E mutant might have
enhanced both the peroxygenation activity and substrate
specificity for the sulfoxidation of 1.
Entry
Enzyme
pH 6.5[b]
pH 8.5
Yield of 2/3 (%)[a]
Yield of 2/3 (%)[a]
1
2
3
4
5
6
7
8
9
10
Wild-Type
T213G
T213M
T213H
F153G
F153A
F153V
F153G/T213G
No. 3–39
K199E
79.1/15.4
74.4/13.0
10.9/–
65.6/38.2
55.4/35.8
39.2/0.36
36.9/0.4
63.3/23.8
59.8/27.3
58.7/32.9
79.2/9.3
14.1/–
80.2[c]/13.5
72.8/10.1
87.7/10.3
90.1/9.4
88.9/8.2
80.4/14.6
P450 119 originated from the organism S. acidocaldarius,
therefor its optimal growth conditions were initially attached to
[6]
82.8/6.5
79.3/16.0
thermophilic and acidophilic sulfur-containing surrounding. It
was found that several natural P450 peroxygenases can catalyze
[a] yield of 2 and 3 determined by GC-MS, [b] concentration of octanoic
acid was 20 mM, [c] enantioselectivity 30% ee determined by HPLC and R-
configuration determined by comparing the HPLC data with the literature
21.
non-native substrates in the presence of short-alkyl-chain
[24]
carboxylic acids.
We thus investigated the sulfoxidation of 1
catalyzed by P450 119 in presence of TBHP and short-alkyl-
chain carboxylic acids. As shown in Table 3, acetic acid was
Table 3. The results of the sulfur oxidation of thioanisole catalyzed by
P450 119 in presence of 20 mM TBHP and various carboxylic acids.
no effect on the sulfur oxidation of 1 catalyzed by the T213G,
T213 M and T213H mutants, in which the 2 and 3 yields were
maintained in comparison with that without any carboxylic acid
(Entries 2–4). The F153G mutation increased the peroxygenation
activity and substrate specificity with a 2/3 yield ratio of 80.2/
13.5 (Entry 5), while the F153 A and F153 V mutants significantly
increased the 2/3 yield ratio to 72.8/10.1 and 87.7/10.3 in the
presence of octanoic acid (Entries 6–7). Especially, the F153G/
T213G mutant showed excellent peroxygenation activity and
substrate specificity for the sulfoxidation of 1 in the presence of
octanoic acid to give the 2/3 yield ratio up to 90.1% versus
9.4% (Entry 8). Octanoic acid was also found to induce a
preferred sulfoxidation of 1 catalyzed by the quadruple mutant
and the K199E mutant with large 2/3 yield ratios of 88.9/8.2 and
80.4/14.6 respectively (Entries 9–10). The sulfoxidation of 1
catalyzed by the P450 119 and its mutants gave R-sulfoxide 2 in
presence of octanoic acid, and the F153G mutant showed
Entry
Carboxylic acid
Yield (%)
ee (%)
Conf.
2/3 [a]
2[b]
2 [c]
1
2
3
4
5
6
none
59.3/30.3
45.5/49.4
53.6/28.9
79.1/15.4
47.8/37.9
47.3/40.9
16.9
23.6
28.4
21.4
28.7
22.1
R
R
R
R
R
R
CH3COOH[d]
CH3(CH2)5COOH[d]
CH3(CH2)6COOH[d]
Ph(CH2)2COOH[d]
Ph(CH2)3COOH[d]
[a] yield of 2 and 3 determined by GC-MS . [b] enantioselectivity
determined by HPLC. [c] configuration determined by comparing the HPLC
data with the literature 21. [d] concentration of carboxylic acid was 20 mM.
found to induce almost equivalent sulfoxidation and sulfona-
tion catalyzed by P450 119 with the yield of 45.5% and 49.4%
respectively (Entry 2), while heptanoic acid gave the yields of 2
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enantioselectivity up to 30% ee (Table S1 in SI). Kinetic
constants were calculated from the resulting regression equa-
tion for the sulfoxidation of 1 catalyzed by the F153G/T213G
mutant in the presence of TBHP and octanoic acid. The T213G/
F153G mutant converted 1 into the sulfoxide 2 with a kcat value
of 3687 minÀ 1 on the basis of the double-reciprocal plot in the
presence of TBHP with octanoic acid (Figure S6 in SI), which
exhibited approximately 2.4-fold increase in turnover rate
relative to that of the wild-type without any carboxylic acid
with a kcat value of 1549 minÀ 1.
To further understand the enhanced activity and stereo-
chemistry in the peroxygenation of P450 119, we also modelled
the docking pose of thioanisole into the active pocket of the
F153G mutant without and with octanoic acid. As shown in
Figure 2a and 2b, the distances between the sulfur atom and
82.8/6.5 and 79.3/16.0 respectively, in which the acidic Glu199
residue with a carboxylic side chain in the mutational K199E
position might play a very important role as a substitution of
octanoic acid. Thus, octanoic acid might enter the catalytic
pocket of P450 119, participate in the formation of Compound
I, and regulate the spatial orientation of thioanisole.
1
2
3
4
5
6
7
8
9
We have proposed a mechanism for the sulfoxidation of 1
catalyzed by the P450 119 F153G peroxygeanse in the presence
of TBHP and octanoic acid according to the studies reported
earlier.[17–18,24–25] As shown in Figure 3, after octanoic acid and
thioanisole enter the catalytic pocket of the P450 119 F153G
peroxygenase, thioanisole could be oriented properly by the
mutant and octanoic acid. The mutant, thioanisole and octanoic
acid together had great influence on the stability of the FeIII
(tBuOOH) intermediate (II) by the hydrogen-bonding interac-
tions in the active site, in which the orientation of thioanisole
could also prevent tBuOOH release. The hydrogen-bonding
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t
network activated by octanoic acid could lock the BuO· radical
formed by the OÀ O homolysis (III), then the radical abstracts
t
the hydrogen atom from FeÀ OÀ H to generate BuOH. Com-
pound I could be formed thereby, which could subsequently
oxidize thioanisole to form the methyl-phenyl sulfoxide 2 with
R-enantioselectivity.
Figure 2. The molecular docking poses of thioanisole 1 into the F153G
mutant (a) without octanoic acid (b) with octanoic acid.
3. Conclusions
We found for the first time that P450 119 peroxygenase
catalyzes the sulfoxidation of thioanisole 1 and the subsequent
sulfonation in the presence of TBHP with turnover rates of
1549 minÀ 1 and 196 minÀ 1 respectively. We rationally designed
several new site-directed P450 mutants, and found that the
F153G/T213G mutant can enhance the peroxygenation activity
and substrate specificity with an increase of the sulfoxide 2
yield and a decrease of the sulfone 3 yield, and the No.3–39
mutant and the K199E mutant with an acidic Glu residue can
the iron-oxo species of the heme are 2.5 Å and 1.8 Å without
and with octanoic acid respectively, which suggests that the
thioanisole molecule corresponding to the formation of the R-
enantiomer of sulfoxide should be closer to the iron-oxo species
of the heme in the active center with octanoic acid than
without octanoic acid.
It was reported that the P450 119 peroxidase activity was
pH dependent in the presence of peroxides, and the peroxidase
[13]
activity was highest at pH 8.5.
We further investigated the
sulfur oxidation of 1 catalyzed by P450 119 and its mutants at
pH 8.5 in presence of TBHP without octanoic acid in order to
confirm the role of the carboxylic acid. As shown in Table 4, the
2/3 yield ratios by using the wild-type (65.6/38.2) and the
T213G mutant (55.4/35.8) at pH 8.5 in the control group were
apparently lower than that at pH 6.5 in the octanoic acid-
attached P450 119 group (Entries 1–2). The T213 M and T213H
mutants gave an enhanced activity for the sulfoxidation of 1
with the yield of the 2 in 39.2% and 36.9% respectively at
pH 8.5, which was in agreement with the P450 119 peroxidase
activity at pH 8.5 reported earlier (Entries 3–4).[13] The F153G,
F153 A and F153H mutations decreased the peroxygenation
activity and substrate specificity with the 2/3 yield ratios of
63.3/23.8, 59.8/27.3 and 58.7/32.9 respectively (Entrie 5–7). The
F153G/T213G mutant also gave an obvious decrease in the 2/3
yield ratio with 79.2/9.3 at pH 8.5 in comparison with that at
pH 6.5 in presence of octanoic acid. The No.3–39 mutant and
the K199E mutant almost maintained the peroxygenation
activity and substrate specificity with more tolerance of the
basic surrounding at pH 8.5, and gave the 2/3 yield ratios of
Figure 3. A proposed mechanism for the sulfoxidation of thioanisole 1
catalyzed by the P450 119 F153G peroxygenase.
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use of the molar extinction coefficient of ɛ415 =104 mM.cmÀ 1. The
reduced-CO complex formation of CYP 119 mutants was monitored
by UV-visible spectroscopy.
improve the 2/3 yield ratio. We investigated the addition of
short-alkyl-chain carboxylic acids into the P450 119 peroxyge-
nase-catalyzed sulfur oxidation of 1, and found that octanoic
acid can induce a preferred sulfoxidation of 1 catalyzed by the
F153G/T213G mutant to give approximately 2.4-fold increase in
turnover rate with a kcat value of 3687 minÀ 1 relative to that of
the wild-type, and by the F153G mutant to give the R-sulfoxide
2 up to 30%ee. The mechanistic study for the P450 119
peroxygenase-catalyzed sulfoxidation of 1 in the presence of
octanoic acid suggests that introduction of the Gly153 residue
into the active pocket of the P450 119 peroxygenase should
contribute to improving the substrate specificity, and octanoic
acid could occupy the catalytic pocket to efficiently participate
in the formation of Compound I by hydrogen-bonding inter-
actions which in combination with the mutant could regulate
the spatial orientation of thioanisole. This is the most effective
catalytic system for the P450 119 peroxygenase-catalyzed
sulfoxidation of thioanisole.
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Synthesis of Standard Samples of the Phenyl methyl
Sulfoxide 2
Hydrogen peroxide (30% by mass, 23.3 mmol) was added dropwise
via addition funnel to a stirred solution of methyl phenyl sulfide
(7.76 mmol) in ethanol (15 mL) on cooled in an ice-water bath. After
the mixture was stirred at RT for 24 h, the reaction mixture was
transferred to CH2Cl2 (50 mL) and cooled in an ice-water bath, and
aqueous sodium bisulfite (15%) was added to decompose the
unreacted H2O2 until the oxidant was no longer visualized by using
the potassium iodide-starch paper. The organic phase was sepa-
rated and dried over Na2SO4 overnight. After filtration, the solvent
was removed by rotary evaporation. After purified by flash
chromatography on a silica gel column, the sulfoxide product was
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1
1
identified by H NMR analysis: H NMR (400 MHz, Chloroform-d) δ
7.66–7.64 (t, 2H), 7.53–7.50 (m, 3H), 2.72 (s, 3H). The sulfoxide
product was used as the standards in the HPLC and GC-MS analysis
for identification of the enzymatic reaction product.
Experimental Section
Synthesis of Standard Sample of Phenyl methyl Sulfone 3
Materials
The reaction was performed in de-ionized H2O (10 mL) containing
methyl phenyl sulfide (2 mmol) and Oxone (3 mmol). The reaction
Escherichia coli strain BL21 (DE3) plysS and pET30a vector were
obtained from Novagen (La Jolla, CA). All restriction enzymes and
ExTaq Polymerase were purchased from TaKaRa Biotechnology
(Liaoning China). Water was generated by using a Milli-Q-Gradient
purification system (Millipore). Medium components tryptone and
yeast extract were purchased from Oxoid. Isopropylb-d-1-thiogalac-
topyranoside (ITPG, >99%), ampicillin sodium salt (>99%),
kanamycin sulfate (>99%), and meta-chloroperbenzoic acid were
purchased from Sigma-Aldrich. Thioanisole was purchased from
J&K Scientific and used without further purification. Acetic acid,
hexanoic acid, heptanoic acid, octanoic acid, 2-phenylpropionic
acid, n-butanoicacid, oxone, hydrogen peroxide were purchased
form Adamas-beta. All other chemicals and reagents were
purchased from J&K Scientific, China.
°
was initiated by vigorous stirring at 60 C for 12 h. Samples were
then extracted with CH2Cl2 (2×2.5 mL) and the combined CH2Cl2
layers were separated and dried over with Na2SO4.The crude
product was purified by flash chromatography on a silica gel
1
1
column and characterized by H NMR analysis: H NMR (400 MHz,
Chloroform-d) δ 7.91–7.88 (m, 2H), 7.62–7.59 (t, 1H), 7.54–7.50 (m,
2H), 3.00 (s, 3H). The sulfone product was used as standards in the
HPLC and GC-MS analysis for identification of the enzymatic
reaction product.
Catalytic Activity Assay
The reaction was carried out in closed glass vials in total volumes of
200 μL containing 7 μM CYP 119 enzyme, 2 mM thioanisole, and
variable TBHP concentrations (for the determination of the
optimum concentration of TBHP) in 50 mM potassium phosphate
Construction, Expression and Purification of CYP119 Mutants
°
buffer, pH 7.5 or in 50 mM glycine buffer, pH 8.5 at 35 C for 1 h. For
Mutants of CYP119 were constructed by using the Quickchange
Lighting Site-directed Mutagenesis Kit (Agilent Techologies). Muta-
genic PCR was applied on pET30a-CYP 119 plasmid constructed
previously as the template DNA.[15] For the T213H mutant, the
forward and reverse primers were 5’-TTCTCATAGCGGGTAATGAG-
CATACAACTAACTTA ATATC-3’, 5’-TTTGATATTAAGTTAGTTGTATGCT-
CATTACCCGCTATGA G À 3’. For the F153 A mutant, the primers
the determination of the short-alkyl-chain carboxylic acids effect, a
concentration of carboxylic acid was 20 mM in 200uL reaction
mixture. Acetophenone was added as an internal standard after the
reaction was quenched with 200 μL CH2Cl2 or hexane. For the
determination of product yield, the reaction mixture was extracted
with 200 μL CH2Cl2 for three times. The CH2Cl2 layer was analyzed
with gas chromatography (Thermo Scientific ITQ900) on a DB-1MS
were
5’-TAGTCGCAGCAAGGTTGGGTA
AGCCTGGAG-3’,
5’-
°
column (30 m×0.25 mm inner diameter, 90 C for 2 min, followed
CAACCTTGCTGCGACTAAGTCTGACCACTC-3’. For the F153 V mutant,
the primers were 5’-AGTCGCAGTTAGG TTGGGTAAGCCTGGAG-3’, 5’-
CCAACCTAACTGCGACTAAGTCTGA CCACTC-3’. For the F153G mu-
tant, the primers were 5’-AGTCGCA GGTAGGTTGGGTAAGCCTGGAG-
3’, 5’-CCAACCTACC TGCGACTAA GTCTGACCACTC-3’. For the K199E
mutant, the primers were 5’- CATAGAGGAACTCGGATACATTATTT-
TACTTCTCA-3’, 5’-TGTATC CGAGTTCCTCTATGTCTGAGAGGTTTGAG-
3’ (Bold and lined face indicates the positions of the mutations). All
enzymes were overexpressed in E.coli BL21 (DE3) plysS cells,
induced by isopropyl-β-D-thiogalactopyranoside as previous study.
°
°
°
by 10 C/min rise to 210 C, and finally 210 C for 1 min). For the
determination of sulfoxide enantioselectivity, the reaction mixture
was then extracted with 200 μL of hexane for three times and the
products were analyzed by using chiral HPLC on a Waters1525
Breeze™ with a UV detector at 254 nm using Daicel Chiralpak
ODÀ H column (hexane/2-propanol=92/8,
tR1 =10.8 min, tR2 =
12.5 min). The configuration of sulfoxide was determined by
comparing the HPLC data with the literature. [21] All the experiments
were carried out in triplicate at least. One control experiment was
done without CYP119 or TBHP. Another experimental control was
conducted for the oxidation of sulfoxide as substrate catalyzed by
P450 119 and TBHP respectively.
[15]
The purity of the CYP119 mutants was determined by SDS-
polyacryl-amide gel electrophoresis with single 45-kDa band eluted
from the Ni-NTA column. Protein concentration was calculated with
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Kinetics Studies
Acknowledgements
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The kinetics constants of P450 119 and its mutants were
determined with thioanisole 1 and sulfoxide 2 as substrate. The
reaction mixture consisted of 0.5 μM CYP119 mutant, variable
concentrations of substrate and TBHP (10-fold concentration of
substrate). Phosphate buffer (pH 7.5) was added to the final volume
This project is supported by Sichuan Science and Technology
Program (2019JDTD0016) and Program of Education Department
of Sichuan Province (14TD0017). We are thankful to Dr. Qin Xu
(School of Life Sciences and Biotechnology, Shanghai Jiao Tong
University, Shanghai, China) for providing Gromacs package
v.5.0.5 for molecular dynamics simulation.
°
at 100 μL. The reaction mixtures were incubated at 35 C for 15
second (for the substrate thioanisole) or 30 second (for the
substrate phenyl methyl sulfoxide) in a closed glass vials and
stopped by addition of CH2Cl2 (100 μL). The products were analyzed
by gas chromatography (Thermo Scientific ITQ900) on a DB-1MS
column (30 m ×0.25 mm inner diameter). The purified sulfoxide 2
and sulfone 3 were used as authentic standards to identify the
retention time and to prepare calibration curve. The retention times
found with this condition were ca. 11.2 min for sulfoxide and ca.
4.5 min for sulfone. The initial rates of formation of sulfoxide and
sulfone were estimated at various concentrations of the substrate
and analyzed by double-reciprocal plot method to evaluate the
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: P450 peroxygenase · site-directed mutagenesis ·
sulfoxidation reactions · turnover rate · catalytic mechanism
kinetic parameters Km and kcat
.
Molecular Dynamics Simulations
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The crystallographic structure of CYP119 complexed with 4-Phenyl-
imidazole (PDB code: 1F4T) was used as the starting point for this
work. The simulation was set up by removing the 4-Phenyl-
imidazole and water molecules. The site directed mutations on the
site 153 were constructed by MOE v2014.0901. All MD simulations
were performed using the Gromacs package v.5.0.5 and the
CHARMM27 all-atom force field. The SPC/E model was used for
water molecules. The protein model was solvated in a rhombic
dodecahedral box, considering a minimum distance between the
protein and box walls of 1 nm. The water molecules and five extra
Na+ ions were added to neutralize the system. The system was
then minimized for 1000 steps, using the steepest descent
algorithm to relieve bad crystal contacts and equilibrated for 400 ps
at 308 K using the NVT ensemble and 105 Pa using the NPT
ensemble. MD simulations were performed for 100 ns with periodic
boundary conditions. The flexibility of the protein was analyzed by
inspecting the root-mean-square fluctuation (RMSF) of each residue
from its time-average position.
Molecular Docking Studies
The crystallographic structure of CYP119 with a resolution of 1.93 Å
was obtained from Protein Data Bank database (PDB code: 1F4T).
The structures of the F135G and F135G/T213G mutants were built
with Swiss-Model (https://www.swissmodel.expasy.org/) by using
the structure of CYP119 as template. Molecular docking study was
carried out with AutoDockTools. The structure of thioanisole was
downloaded from PubChemCompound database. Before docking
simulation, solvent was removed from the protein structures. Heme
and oxygen atom were retained and hydrogen atoms were added.
The substrate thioanisole was treated as a flexible ligand and
allowed to rotate freely. Autogrid (4.2) was used to generate grid
maps for the ligand and the grid box was fixed to cover the
catalytic site containing heme moiety. Local Search Parameters
were selected and Docking Parameters were set by default in the
docking process with Autodock (4.2) program. The docking results
were analyzed by using Pymol.
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2007, 119, 3730–3733.
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Fornarini, M. E. Crestoni, S. P. De Visser, Chem. Sci., 2015, 6, 1516–1529;
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FULL PAPERS
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X. Wei, Dr. C. Zhang, Dr. X. Gao, Y.
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Gao, Y. Yang, Dr. K. Guo, X. Du, L.
Pu*, Prof. Dr. Q. Wang*
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Enhanced Activity and Substrate
Specificity by Site-Directed Muta-
genesis for the P450 119 Peroxy-
genase Catalyzed Sulfoxidation of
Thioanisole
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New mutants: The P450 119 T213G/
F153G peroxygenase converted thioa-
nisole into the sulfoxide with a kcat
value of 3687 minÀ 1 in the presence of
TBHP with octanoic acid.