Engineering P450LaMO stereospecificity and product selectivity for selective C-H oxidation of tetralin-like alkylbenzenes

Article abstract of DOI:10.1039/c8cy01448e

The P450-mediated asymmetric hydroxylation of inert C-H bonds is a chemically challenging reaction. Self-sufficient P450_LaMO from the CYP116B subfamily could catalyze the transformation of 1,2,3,4-tetrahydronaphthalene to (S)-tetralol, despite its poor enantioselectivity (er 66:34) and product selectivity (the ratio of alcohol and ketone, ak, 76:24). To improve the selectivity, phenylalanine scanning and further protein engineering were performed to reshape the active pocket of P450_LaMO, resulting in a mutant (T121V/Y385F/M391L) with not only improved (S)-enantioselectivity (er 98:2) but also excellent product selectivity (ak 99:1), in contrast to another mutant L97F/T121F/E282V/T283Y with complementary (R)-enantioselectivity (er 23:77). Moreover, the enantiopure (S)-alcohols formed by the P450_LaMO-catalyzed oxidation of a series of alkylbenzenes are potentially important building blocks in the pharmaceutical industry. This Phe-based enantioselectivity engineering used for reshaping the active pocket of P450s could provide a guide to the protein evolution of other CYP116B members.

Full text of DOI:10.1039/c8cy01448e

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Engineering P450LaMO Stereospecificity and Product Selectivity for
Selective C-H Oxidation of Tetralin-like Alkylbenzenes
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
c
a
d
a
a
Ren-Jie Li, Aitao Li, Jing Zhao, Qi Chen, Ning Li, Hui-Lei Yu * and Jian-He Xu
DOI: 10.1039/x0xx00000x
P450-mediated asymmetric hydroxylation of inert C-H bond is a chemically challenging reaction. Self-sufficient P450LaMO
from CYP116B subfamily could catalyze the transformation of 1,2,3,4-tetrahydronaphthalene to (S)-tetralol, despite with
poor enantioselectivity (er 66:34) and product selectivity (the ratio of alcohol and ketone, ak, 76:24). To improve the
selectivity, phenyalanine scanning and further protein engineering were performed to reshape the active pocket of
P450LaMO, resulting in a mutant (T121V/Y385F/M391L) with not only improved (S)-enantioselectivity (er 98:2) but also
excellent product selectivity (ak 99:1), in contrast to another mutant L97F/T121F/E282V/T283Y with complementary (R)-
enantioselectivity (er 23:77). Moverover, the enantiopure (S)-alcohols formed by P450LaMO-catalyzed oxidation for a series
of alkylbenzenes are potentially important building blocks in the pharmaceutical industry. This Phe-based
enantioselectivity engineering used for reshaping the active pocket of P450s could provide a guide to the protein evolution
www.rsc.org/
of
other
CYP116B
members.
monooxygenase, contains novel FMN and Fe
2
S
2
domains for
1
2
Introduction
conducting electrons transfer to the heme domain. This type
of P450 monooxygenases are reported to be able to perform
Biocatalysis plays an important role in green and sustainable
synthesis of enantiopure compounds, especially chiral
1
3
14
C-H hydroxylation, O-demethylation, styrene epoxidation
5
1
5
1
alcohols. The asymmetric hydroxylation of inert C-H bonds
and sulfide oxidation. Recently, it was reported that P450LaMO
could be used for anti-Markovnikov addition of styrene
derivatives, with an improved activity and selectivity after
could be achieved by traditional chemical methods, but they
2
are generally costly, energy-intensive and non-selective. The
1
1
directed evolution.
It is known that (S)-tetralol can be used as
P450 (Cytochrome P450)-mediated asymmetric hydroxylation
of inert C-H bond is of great interest and has a great potential
a
key
3
intermediate for synthesis of bioactive compounds, such as
prodrugs of meropenem, JAK3 inhibitor and ORL-1 receptor
to form highly enantiopure hydroxylated products.
P450s catalyze various chemically challenging reactions,
such as non-activated C-H hydroxylation, alkene epoxidation
1
agonist. Whereas very few reports are available regarding
6
1
7
4
and sulfoxidation. The typical representatives (e.g. P450cam,
5
P450 monooxygenase catalyzed production of (S)-tetralol,
several of the self-sufficient CYP116B members, including
were employed for
6
7
P450pyr and P450BM3 ) displayed high activity and excellent
regio-/enantioselectivity after being tailored by molecular
P450RpMO, P450CtMO and P450ArMO,
asymmetric oxidation of the core structure of tetralin (1a) into
14b
tetralol (2a). Particularly, the native P450LaMO was able to
engineering approaches. For example, the evolved P450
BM3
8
variants show impressive promiscuous activity and can be
9
used for obtaining the products of carbene and nitrene
convert 1a into (S)-2a despite of the poor enantioselectivity (er
66:34, S/R) and product selectivity (alcohol : ketone, ak 76:24).
Therefore, this research was focused on the directed evolution
of P450LaMO for producing (S)-tetralol and other chiral alcohols
1
transfer with high enantioselectivity. Another self-sufficient
0
1
1
CYP116B member, P450LaMO
,
belonging to Class VII
(Scheme 1) with better product selectivity and
enantioselectivity.
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thiogalactopyranoside) and 0.2 mM ALA (5-aminolevulinic acid
hydrochloride) were added to each well and the temperature
was decreased to 16 °C for incubation for another 24 h. The
cells were then harvested by centrifugation (3220 × g, 10 min).
The cell pellets were resuspended in 300 µL potassium
phosphate buffer (100 mM, pH 8.0, saturated with oxygen)
and transferred into 96-well microplates.
The reaction mixture (150 µL), containing whole cells
expressing P450LaMO (1.6
g CDW/L), 1.0 mM 1,2,3,4-
tetrahydronaphthalene, and 100 mM KPi buffer (pH 8.0) in 96-
deep well plates, was incubated at 220 rpm for 5 h. Then, the
mixture was separated by centrifuge (3220 × g, 15 min) and
1
30 µL supernatant was dispensed into two new microplates
to perform further dehydrogenation. The dehydrogenation
was started by separately adding 10 µL CpCR (0.5 U) and 10 µL
mixed NBT-PMS solution (NBT 2 mg/mL, PMS 0.1 mg/mL and
Scheme
1 Cytochrome P450LaMO catalysed asymmetric
hydroxylation of alkylbenzenes to form corresponding (S)-
+
NADP 0.3 mM), and then the change in absorbance at 580 nm
alcohols.
was monitored after the reaction was kept at 30 °C for 1 h in
the dark (Fig. S1† and Table S5†).
Thus far, various approaches have been proffered on
1
8
reshaping the active pocket of enzymes to improve the
enantioselectivity, such as single codon saturation
mutagenesis (SCSM), double codon saturation mutagenesis
Measurement of Total Turnover Number and Turnover
frequency
1
8i,19
(DCSM) and triple codon saturation mutagenesis (TCSM).
All of the enzymatic transformations were performed in 1-mL
However, there was no systematical study on probing the reaction tubes on a mini-shaker (HLCBioTech, MHR23) at 600
active pocket of P450LaMO to enhance its enantioselectivity. rpm, 20 °C, 5 min. The reaction mixtures (1 mL) were
2
1
Herein, we tried to improve the stereoselectivity and product composed of 0.2 µM P450s, 0.5 mM NADPH and 0.5 mM
selectivity through phenylalanine (Phe)-based scanning to substrate. The total turnover number (TTN) of reactions were
reshape the active pocket, followed by polarity optimization of measured at 1-mL scale in 5 mL crimp vials using oxygen
active pocket with valine.
saturated KPi buffer (100 mM, pH 8.0) containing 0.8 µM
+
purified P450, 5 mM substrate, 0.5 mM NADP , 15 U/mL
glucose dehydrogenase, and 20 mM glucose. The vials were
sealed and stirred for 12 h at 25 °C and 180 rpm. Then 500 µL
ethyl acetate was used (with 0.5 mM guaiacol as an internal
standard) to extract the sample twice, and the organic layers
Experimental
Materials
1,2,3,4-Tetrahydronaphthalene and alkylbenzenes compounds
2 4
were combined and dried over anhydrous Na SO .
and the corresponding racemic alcohol standards used in this
work were of analytical grade and commercially available. δ-
Aminolevulinic acid hydrochloride, isopropyl β-D-1-
thiogalactopyranoside (IPTG, >99%) and kanamycin disulfate
Subsequently, the extract was concentrated to 50 µL to
perform GC-MS analysis for quantitation of the reaction
product. For more details, see the ESI.
salt (>99%) was from Sangon (Shanghai, China). PrimeSTAR HS Molecular Docking and Mutation Site Exhibition
DNA polymerase was obtained from TaKaRa (Shanghai, China).
A structural model of P450LaMO wildtype was constructed
DpnI was purchased from New England Biolabs (Beverley, MA).
based on the crystal structures of its homologue enzyme
Plasmid pRSFDuet1 was obtained from Novagen (Madison, WI).
P450TT (CYP116B46) (PDB ID: 6GII, 1.9
Å, 55% identity to
Tryptone and yeast extract were purchased from OXOID
Shanghai, China), NaCl, K HPO , KH PO were commercially
P450LaMO). The online SWISS-MODEL web server
(http://www.swissmodel. expasy.org/) was used to perform
the homology modeling (the assessment of the homologous
(
2
4
2
4
available. Escherichia coli competent cells BL21 (DE3) were
2
0
prepared using a standard protocol. Sequence analysis was
performed by Sain biological (Shanghai, China).
22
modelling was shown in Fig S8, ESI). The 3D structure of
substrate tetralin was generated by energy minimization. Then
High throughput screening process for enantioselectivity the substrate structure was docked into the binding pocket of
evolution
the constructed P450LaMO model (AutoDock 4.0). The best
scoring results were selected for structural comparison and
catalytic mechanism analysis. After introducing mutagenesis,
the mutation sites were shown by the visual software Pymol
Single colonies were picked with toothpicks, inoculated and
grown at 37 °C in 96-deep well plates with 300 µL LB medium
per well, containing 50 mg/L kanamycin, using wildtype as the
positive control. The expression culture was inoculated by
2.5.
transferring 50 µL overnight culture into 650 µL Terrific Broth Preparative Hydroxylation Reaction
medium supplemented with 50 mg/L kanamycin. When the
The preparative hydroxylation reactions were performed
OD600 reached ~1.0, 0.2 mM IPTG (isopropyl-β-D-
according to the earlier stated procedure: The 125-mL scale
2
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reaction was performed in a 500-mL grinding mouth reactor, is considered to be more liable for reshaping the active pocket.
2
containing 2 µM P450 (purified enzymes), 20 mM glucose, 4 Among the resultant 5 mutants, RJ1 (T121F) showed improved
1
mM substrate, 15 U glucose dehydrogenase, and 0.5 mM (S)-enantioselectivity (er 93:7). Interestingly, another mutant
+
NADP in KPi buffer (100 mM, pH 8.0). The reaction was RJ4 (L97F) inverted the enantioselectivity, forming (R)-product
carried out at 25 °C with magnetic stirring for 12 h. After with an er of 47:53 (Step 1 in Scheme 2). The mutation at the
completion, the reaction mixture was extracted with ethyl remaining three sites did not affect the enantioselectivity,
acetate (100 mL × 2) and the organic phase was dried, giving similar er values with that of the wildtype. Subsequently,
concentrated and subjected to gas chromatography to analyze two rounds of NNK-based saturation mutagenesis were
the product formation.
performed at other four (Y385, M391, M326, V327, Fig. 1B)
sites surrounding the heme, based on iterative saturation
mutagenesis (ISM) of variant RJ1 (Step 2 in Scheme 2). The
selectivity screening was conducted by a colorimetric assay of
dehydrogenase coupled with NBT-PMS (Fig. S1† and Table S5†).
The resulting mutant RJ2 (T121F/Y385F/M391L) further
improved the er to 95:5 and ak to 95:5, enhancing the TOF and
TTN by 4.3-fold and 7.1-fold over WT, respectively. In order to
further enhance the selectivity, we optimized the polarity and
hindrance by single-site mutagenesis (SSM) on residue T121 of
RJ2, resulting in mutant RJ3 (T121V/Y385F/M329L, Step 3 in
Scheme 2). It gave the highest enantioselectivity with an er of
up to 98:2 (S/R), and almost no side product was detected (ak
Analytical instruments and methods
Analytical high-performance liquid chromatography (HPLC)
was performed with Shimadzu 2010A Infinity instrument using
an OD-H column (Daicel, 4.6 × 5 mm, 2.7 µm) with n-hexane
and isopropanol as the mobile phase to analyze the
enantioselectivity. All measurements were performed with a
Shimadzu 2010 Infinity Diode Array Detector and the recorded
wavelength was set at 254 nm.
Gas chromatography (GC) analyses were carried out with a
Shimadzu GC-2014 instrument equipped with
ionization detector using an Agilent Rsi-MS column (30 m ×
.32 mm, 0.25 µm film) with nitrogen as carrier gas to analyze
a flame
0
9
9:1, Fig. S3† and Fig. S4†). Furthermore, we found that
the product distribution. Injector temperature: 280 °C. Split
mode with a split ratio of 10. Detector temperature: 280 °C.
Chiral GC analysis was conducted with an Agilent 7820A
instrument equipped with a flame ionization detector using a
chiral Agilent CP column (30 m × 0.32 mm, 0.25 µm film) with
helium as carrier gas to analyze the configuration of products.
Injector temperature: 200 °C. Split mode with a split ratio of 5.
Detector temperature: 280 °C. The retention time of all the
mutant RJ33 (T121P/Y385F/M329L, Table S4†) displayed
higher activity after polarity optimization on site T121 in the
active pocket in spite of a slightly decreased er (94:6).
1
1a
compounds see the ESI.
NMR data
Two products 2a and 2d were selected to perform the NMR
analysis. Another four products’ configuration confirmed by
1
1a
the previous work, NMR spectra see ESI. More details are as
follows:
1
Data for 2a, (S)-1,2,3,4-tetrahydronaphthalen-1-ol. H NMR
(400 MHz, DMSO-d ) δ 7.37-7.33 (m, 1H), 7.13-7.08 (m, 2H),
6
7
.03-7.01 (m, 1H), 5.05-5.03 (br, 1H), 4.56-4.52 (m, 1H), 2.76-
3
1
Scheme 2 Directed evolution pathways producing the (S)- or
6
2.60 (m, 2H), 1.88-1.64 (m, 4H). C NMR (100 MHz, DMSO-d )
(
R)-selective P450LaMO mutants as catalysts for the asymmetric
δ 145.5, 141.6, 133.6, 133.5, 131.8, 130.7, 71.6, 37.5, 34.0,
4.3.
Data for 2d, (S)-1-phenylenthan-1-ol. H NMR (400 MHz
CDCl
hydroxylation of substrate 1a. Step 1: phenylalanine scan. Step
2: iterative saturation mutagenesis. Step 3: single site
saturation mutagenesis. Step 4: combinatorial mutagenesis.
2
1
，
3
): δ = 7.37-7.27 (m, 5H), 4.88 (q, J = 4.0 Hz, 1H), 2.06 (s,
1
3
1
H), 1.48 (d, J = 4.0 Hz, 3H). C NMR (100 MHz, CDCl
3
): δ 145.8,
In Step 1, RJ4 (L97F) inverted the enantioselectivity. To
further improve the (R)-enantioselectivity, double site semi-
saturation mutagenesis (with NDT codon degeneracy) was
1
28.6, 127.5, 125.4, 70.4, 25.2.
1
8a,23
Results and discussion
carried out based on RJ4 (L97F).
About 1600
randomization variants (with 95% coverage) focusing on sites
L97-K99, H206-W211, E282-T283 and A279-A280 (internal and
To enhance the enantioselectivity, the Phe-substituting
mutagenesis at five selected residues (L97, T121, V123, N124
and A275) lining the active pocket of P450LaMO was firstly
2
4
outside active pocket, Fig. 1B) were screened. However, the
R)-selectivity was not obviously enhanced and the resulting
mutant RJ5 (L97F/E282V/T283Y) yielded the (R)-alcohol with er
1
8i,19f
(
performed (Fig. 1A).
Instead of mutating these sites by the
single site saturation mutagenesis (with NNK codon
degeneracy), Phe scan was employed based on its non-polarity,
larger hindrance and hydrophobic side chain properties, which
4
5:55 and ak 91:9 (Step 2 in Scheme 2, Table S6†). Moreover,
the catalytic efficiency was promoted, with increases of TOF
and TTN by 1.8-fold and 2.4-fold, respectively. When
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introducing the mutation of T121F into variant RJ5, the
During the characterization of variants, we found that pH
resulting variant RJ6 (L97F/T121F/E282V/T283Y, Step 4 in 7.4 of the working solution and ethanol as a cosolvent (<5%,
Scheme 2) improved the (R)-selectivity from er 45:55 (S/R) to v/v) increased the enantioselectivity and product selectivity,
-
1
er 23:77, although the activity (TOF: 0.02 min ) was reduced respectively (Fig. S5† and Fig. S6†). It was shown that the
-
1
by half compared to wildtype (TOF: 0.04 min ) (Table S6†).
environmental conditions of P450s may affect the dynamic
conformation of holoenzyme, leading to the changes in
selectivity. Moreover, the E. coli cells harboring an empty
plasmid could not transform the tetralol into tetralone which
excluded another factor affecting the product selectivity (Fig.
S7†).
Some of the P450LaMO mutants engineered for hydroxylation
of 1a were characterized by measuring the turnover frequency
-
1
(
Table 1). The mutant RJ3 improved the TOF from 0.04 min
-1
(
WT) to 0.71 min , which represents about 18-fold
improvement. The TTN was enhanced from 116 to 1360, which
corresponds to a 12-fold improvement. For the mutant RJ33,
compared with the wildtype, about 18-fold improvement on
TTN (from 116 to 2104) and 21-fold enhancement on TOF
-
1
-1
(
from 0.04 min to 0.84 min ) were achieved, respectively.
These results show that this strategy is effective for improving
not only the enantioselectivity but also the catalytic activity.
Moreover, all the mutants maintained the absolute
regioselectivity toward 1-position, without forming any other
product except the ketone (Fig. S3† and Table S4†).
To explore the substrate profile, the best mutant RJ33 was
employed to transform several other alkylbenzene compounds
(
1b-1e). The measured enantioselectivity and product
selectivity are presented in Table 2. It shows that moderate to
high degrees of enantioselectivity and product selectivity were
Fig. 1 Selected amino acid residues in the active pocket of achieved. In addition, the transformation of these substrates
P450LaMO for protein engineering. A) Selected residues lining by P450LaMO gave better enantioselective (S)-product.
In order to provide insights into the (S)-enantioselective
the active pocket were used for the Phe scan. B) Selected
residues were subjected to saturation mutagenesis on either hydroxylation of P450LaMO, we performed in silico modelling of
single site or double sites shown in different areas: the monooxygenase, and docked substrate 1a into the
surrounding the heme ring (pink), internal acive pocket (green), modelled structures of wildtype and mutant RJ3 (Fig. 2). In the
outside active pocket (blue).
substrate
T121V/Y385F/M391L), the average distances between the
substrate C-H bond and the center of heme were 4.20 Å
average 4.7 Å and 3.7 Å) and 3.95 Å, respectively (Fig. 2A and
binding
poses
of
wildtype
and
RJ3
(
To probe the catalytic mechanism of wildtype and mutants,
the kinetic isotope effects (KIEs) was measured, giving KIE
values of 3.1 and 2.8/2.7 for WT and (S)-/(R)-selective mutants,
(
2
5
Fig. 2B). Moreover, mutation T121V gave a hydrophobic
residue Val121 which improves the hydrophobic effect in the
active pocket. It cooperates with other two residues F385 and
L391 around heme to provide a hydrophobic environment
which is more liable for conducting hydroxylation of non-polar
substrates. To get the clues for improvements of the
enantioselectivity, (S)-tetralol was docked into the modelled
structures of wildtype and mutants, respectively. The hydroxyl
group was formed on the position of C-H bonds by both
wildtype and mutant RJ3 (Fig. 2C and Fig. 2D). The mutant RJ3
shows a shorter distance between O- atom of (S)-hydroxy and
the center of heme after reshaping the active pocket (from 3.6
to 2.6 Å), as compared to that of the wildtype (3.7 to 2.9 Å).
Moreover, the hydrophobic effects promote the entering of
substrate into the active pocket in a more appropriate manner
to perform the H abstraction from C-H bonds. These results
show that the mutant RJ3 is more favorable for forming the
respectively (Fig. S2† and Table S7†). It is suggested that
these P450s with different stereopreference still share the
same process of C-H bond breaking.
Table 1 Catalytic performance of wildtype and mutants
evolved for asymmetric hydroxylation of substrate 1a
-1 a
TOF (min )
b
c
Entry P450LaMO
TTN
116
724
852
er
1
2
3
4
5
WT
RJ1
RJ2
RJ3
RJ33
0.04
66:34
93:7
95:5
98:2
95:5
0.23
0.17
0.71
1360
2104
0.84
a
b
TOF: turnover frequency, measured within 5 min. TTN, total
turnover number was calculated after 24 h. More details see
c
the Experimental Section. Enantioselectivity was analyzed by
HPLC, with an OD-H column, ESI. (S)-tetralol was formed for all
entries.
(
S)-tetralol product.
4
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Fig. 2 Molecular docking poses of wildtype and mutant of P450LaMO. (A) Molecular docking pose of wildtype for substrate 1a. (B)
Molecular docking pose of mutant RJ3 for substrate 1a. C) Molecular docking pose of wildtype for (S)-2a. D) Molecular docking
pose of mutant RJ3 docking pose for (S)-2a. All the results were based on in silico mutation of a homology model.
Table 2 Catalytic parameters of variant RJ33 for substrates 1b–1e
11a,26
c
d
Substrate
Structure
P450LaMO
WT
Product
er (S:R)
ak
TTN
333
a
56:44
67:33
83:17
75:25
87:13
92:8
1
b
a
RJ33
WT
92:8
999
133
399
b
86:14
1c
b
RJ33
WT
93:7
b
87:13
1867
5533
267
1
d
e
b
RJ33
WT
96:4
99:1
100
100
b
94:6
b
RJ33
96:4
333
1
b
WT
75:25
93:7
95:5
1067
b
RJ33
91:9
1000
a
b
c
er: enantiometric ratio, analysed by HPLC with OD-H column. Enantioselectivity measured by GC with chiral CP column. ak value: the
d
molar ratio of alcohol over ketone (defined as product selectivity), analysed by GC with Rsi-MS column. TTN, calculated after 24 h. The
reaction mixture (1 mL) was composed of 2 mM substrate, 0.5 mM NADP , 20 mM glucose, 15 U GDH, 0.3 µM P450s (cell free extracts), and
+
1
00 mM potassium phosphate buffer, pH 8.0.
Conclusions
In summary, because of the potential applications of (S)- (KIE) was measured, giving KIE values of 3.1 and 2.8/2.7 for WT
tetralol as biologically active compounds, the biocatalytic and (S)-/(R)-selective mutants, respectively (Fig. S3†, Table
2
5
hydroxylation of 1,2,3,4-tetrahydronaphthalene
attempted utilizing P450LaMO as a catalyst with oxygen as stereopreference still share the same process of C-H bond
oxidant. Since the wildtype enzyme yielded only poor breaking. For alkylbenzene-like C-H substrates (1b 1c 1d 1e),
(1a) was S4†). It is suggested that these P450s with different
,
,
,
enantioselectivity (er 66:34) in favor of (S)-2a and moderate the mutants also enhanced the product selectivity and
product selectivity (ak 76:24), protein engineering was enantioselectivity. The resulting mutants are therefore an
performed through reshaping the active pocket to generate alternative to the chemical catalysts for performing C-H bond
2
7
P450LaMO mutants with increased (S)-selectivity (up to er 98:2) activation. Furthermore, the Phe-based mutation strategy as
as well as mutants showing reversed (R)-selectivity (er 23:77). described in this work may be beneficial for applications in
Meanwhile, the ak value was enhanced up to 99:1 through the future directed evolution of other potential industrial
introduction of key residues (Fig. S2†). To probe the catalytic important enzymes.
mechanism of wildtype and mutants, the kinetic isotope effect
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Nos. 21672063 & 21536004), the
Fundamental Research Fund for the Central Universities (No.
222201514039), and the Science and Technology Commission
of Shanghai Municipality (No. 15JC1400403). We thank Taiwo
Dele-Osibanjo (Tianjin Institute of Industrial Biotechnology) for
discussing the manuscript.
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Via Phe scanning based protein engineering, P450LaMO increased the enantioselectivity to er
8:2, product selectivity, alcohol: ketone to ak 99:1.
9