Significantly enhancing the biocatalytic synthesis of chiral alcohols by semi-rationally engineering an anti-Prelog carbonyl reductase from Acetobacter sp. CCTCC M209061

Article abstract of DOI:10.1016/j.mcat.2019.110613

Chiral alcohols and their derivatives are vital building blocks to synthesize pharmaceutical drugs and high-valued chemicals. Wild-type carbonyl reductase AcCR from Acetobacter sp. has ideal enantioselectivity toward 11 prochiral substrates (e.e.>99%) but poor activity. In this work, a semi-rational engineering was performed to enhance the activity of AcCR. Fortunately, three positive double-mutants (mut-E144A/G152 L, mut-G152 L/Y189 N, and mut-I147 V/G152 L) with specific activity 17–61 folds higher than that of enzyme without modified were achieved. Kinetic studies suggested that the catalytic efficiencies (k_cat/K_m) of these mutants were also well enhanced. Finally, these modified mut-AcCRs were successfully applied in asymmetric reductions of 11 structurally diverse prochiral substrates (200 mM) with excellent product yields (76.8%–99.1%) and enantiomeric excess (e.e.>99%), which provides an alternative strategy for efficient synthesis of chiral alcohols for pharmaceuticals industry with ideal yield and enantioselectivity.

Full text of DOI:10.1016/j.mcat.2019.110613

MolecularCatalysis479(2019)110613
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Significantly enhancing the biocatalytic synthesis of chiral alcohols by semi-
rationally engineering an anti-Prelog carbonyl reductase from Acetobacter
sp. CCTCC M209061
Ping Weia,b,1, Ze-Wang Guoa,1, Xiao-Ling Wua, Shan Lianga, Xiao-Yang Oua, Pei Xua,
⁎
Min-Hua Zonga, Ji-Guo Yangc, Wen-Yong Loua,c,
a Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510641,China
b School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China
c Innovation Center of Bioactive Molecule Development and Application, South China Institute of Collaborative Innovation, Dongguan 221116, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chiral alcohols and their derivatives are vital building blocks to synthesize pharmaceutical drugs and high-
valued chemicals. Wild-type carbonyl reductase AcCR from Acetobacter sp. has ideal enantioselectivity toward 11
prochiral substrates (e.e. > 99%) but poor activity. In this work, a semi-rational engineering was performed to
enhance the activity of AcCR. Fortunately, three positive double-mutants (mut-E144A/G152 L, mut-G152 L/
Y189 N, and mut-I147 V/G152 L) with specific activity 17–61 folds higher than that of enzyme without modified
were achieved. Kinetic studies suggested that the catalytic efficiencies (kcat/Km) of these mutants were also well
enhanced. Finally, these modified mut-AcCRs were successfully applied in asymmetric reductions of 11 struc-
turally diverse prochiral substrates (200 mM) with excellent product yields (76.8%–99.1%) and enantiomeric
excess (e.e. > 99%), which provides an alternative strategy for efficient synthesis of chiral alcohols for phar-
maceuticals industry with ideal yield and enantioselectivity.
Carbonyl reductase
Asymmetric reduction
Semi-rational design
Chiral alcohols
1. Introduction
carbon dioxide in biphasic systems [15,16]. The second challenge is the
limited activities of most carbonyl reductases confined by substrate
Chiral alcohols and their derivatives are important building blocks
that have been widely used in synthesis of various vital chiral chemicals
such as chiral pharmaceuticals, flavors, liquid crystals and biointerface
materials [1–3]. Currently, biosynthesis relies on the enzymes and
whole-cells has been an alternative for production of chiral alcohols
[4–7]. Compared to chemical synthesis, enantioselective carbonyl
compound reductions are reliable, green and straightforward routes to
obtain optically active alcohols [8–10].
Carbonyl reductase (EC 1.1.1.148) as efficient oxidoreductase has
been proved to be powerful biocatalysts for the asymmetric reduction of
carbonyl substrates to the synthesis of chiral alcohols [11–14]. How-
ever, two major challenges restricted the efficiency of biocatalytic
redox reductions of carbonyl reductases for their applications in
asymmetric synthesis. First, most of the interesting substrates are in-
soluble or poorly soluble in the natural enzyme-containing aqueous
phase. This disadvantage has been effectively conquered by using non-
aqueous media like ionic liquids, organic solvents and supercritical
specificity, coenzyme dependence and elasticity of physiological en-
vironment, resulting in the lack of an appointed asymmetric reduction
efficiency [17–19]. For example, an (S)-carbonyl reductase from Can-
dida parapsilosis is only capable to catalyze the reduction of 2-hydroxy-
1-phenylethanone to (S)-1-phenyl-1,2-ethanediol at the conditions of
pH 6.5 and 30 °C [20–23]. Additionally, a carbonyl reductase (NcCR)
from Neuropora crassa showed strict substrate specificity to many kinds
of ketones, with 468 U/mg activity being confirmed in reduction of
dihydroxyacetone. However, the enantioselectivity of NcCR for reduc-
tion of ethyl 4-chloro-3-oxobutanoate strongly depended on tempera-
ture, revealing that the product e.e. value could be dramatically de-
creased by increasing the temperature from −3 °C to 40 °C (98.0% vs
78.8%) [24].
To improve the performance of the carbonyl reductases, various
enzymes have been successfully modified by using random mutagenesis
and rational design to improve operational stability, expand the range
of substrate spectrum, and improve regio-and/orstereo-selectivity
⁎ Corresponding author at: Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China.
E-mail address: wylou@scut.edu.cn (W.-Y. Lou).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.mcat.2019.110613
Received 27 May 2019; Received in revised form 4 August 2019; Accepted 9 September 2019
Availableonline14September2019
2468-8231/©2019ElsevierB.V.Allrightsreserved.

P. Wei, et al.
MolecularCatalysis479(2019)110613
Fig. 1. Catalytic triad and mutation sites.
unsatisfactory [32–34]. Therefore, a semi-rational design based on the
combination of homology modelling and saturation site-directed mu-
tagenesis was applied for the modification of AcCR. Apparent kinetic
parameter and docking experiments were used to illustrate the me-
chanism of the changes in enzyme characteristics. Finally, three posi-
tive double-mutants were obtained with excellent product yields
(76.8%–99.1%) and enantiomeric excess (e.e. > 99%), suggesting its
potential application to product the pharmaceutically relevant chiral
alcohols.
Table 1
Enzyme activity of different AcCR mutants.
Enzyme 4’-Chloroacetophenone
Ethyl 2-oxo-4-
2-Hydroxy-1-
phenylbutyrate (2a) phenylethanone
(3a)
(1a)
Enzyme
activity
[U/mg]a
Fold changeb Enzyme
Fold
Enzyme
Fold
activity
changeb activity
[U/mg]a
changeb
[U/mg]a
AcCR
A94Q
S96Y
5.17
4.07
5.22
5.99
47.24
5.05
5.69
23.19
6.92
5.79
2.03
1.00
0.79
1.01
1.16
9.14
0.98
1.10
4.49
1.34
1.12
0.39
0.58
1.45
2.73
1.61
1.67
0.90
5.98
1.91
5.82
4.50
4.25
73.44
5.39
1.00
1.88
1.11
1.15
0.62
3.62
1.31
4.01
3.10
2.93
50.58
3.72
0.37
0.27
0.42
0.34
0.28
1.76
1.90
4.33
0.83
0.15
0.12
1.37
1.00
0.73
1.13
0.90
0.74
4.71
5.06
11.56
2.22
0.40
0.33
3.66
2. Experimental
S96T
E144A
I147V
M151T
G152L
G152A
G152C
Y189N
2.1. Chemicals, plasmids and strains
BL21(DE3) (Novagen, Darmstadt, Germany) was as the host cell,
and pGEX plasmid was used for cloning and expression. BL21(DE3)/
pGEX-AcCR was used to express AcCR. PrimeSTAR Max DNA poly-
merase was purchased from Takara; FastDigestDpnI was purchased
from Thermo Fisher Scientific. NADH, ethyl 2-oxo-4-phenylbutyrate
(2a), 2-hydroxy-1-phenylethanone (3a) and 1-phenyl-1,2-ethanediol
(3b) were purchased from Aladdin; 4′-chloroacetophenone (1a), 4′-
bromoacetophenone (5a), 3′-methoxyacetophenone (6a), 4′-methox-
yacetophenone (7a), methyl acetoacetate (8a), ethyl acetoacetate (9a),
ethyl-4-chloroacetoacetate (10a), 3-methoxyphenethyl ethanol (6b), 4-
methoxyphenethyl ethanol (7b), 1-(4-chlorophenyl) ethanol (1b), 1-(4-
bromophenyl) ethanol (5b), methyl 3-hydroxybutyrate (8b), ethyl 3-
hydroxybutyrate (9b) and 4-chloro-3-hydroxybutanoate (10b) were
purchased from Sigma Aldrich; acetophenone (11a), phenylethanol
(11b) and ethyl 2-hydroxy-4-phenylbutyrate (2b) were purchased from
TCI (Shanghai) Development Co., Ltd.; 4-fluoroacetophenone (4a) and
1-(4-fluorophenyl) ethanol (4b) were purchased from Alfa Aesar. All
otherchemicals were analytical grade purity.
W191A 2.98
a
0.25 mM NADH, citrate-phosphate buffer (pH 6.5, 50 mM), and 5 mM 1a or
2a or 3a were incubated for 5 min at 35 °C followed by adding a certain amount
of purifed enzyme, the changes in absorbance after 3 min were recorded.
b
Fold change improvement in enzyme activity over the WT enzyme.
[25–29]. For example, a carbonyl reductase from Ogataeaminuta with
improved thermostability was achieved by directed evolution strategy.
As a result, in the absence and presence of 20% (v/v) dimethyl sulf-
oxide, the half-lives of mutant-V166A were 6.1 and 11 times longer
than those of the wild type one at 50 °C, respectively [30]. Additionally,
activity improvement of Kluyveromyces lactis aldo-keto reductase KlAKR
was carried out via rational design. On the basis of homology modeling
and molecular docking, mutant KlAKR-Y295W/W296 L showed the
highest catalytic efficiency (kcat/Km) tot-butyl 6-cyano-(5R)-hydroxy-3-
oxohexanoate, the kcat/Km was up to 12.37 s−1 mM−1, which was 12.5
folds higher than that of the wild type one, showing that the efficient
activity improvement of KlAKR was realized through rational design
successfully [31]. Therefore, protein engineering as a kind of powerful
tool for tailoring enzymes could significantly improve the performance
of the biocatalyst.
2.2. Analytical methods of the substrates and chiral alcohols
GC methods. The samples were analyzed by GC analysis (GC 2010,
Shimadzu Corp). Use chiral columns HP Chiral 10B, HP Chiral 20% or
CP-Chiralsil-Dex-CB (Agilent Technoligies, China). The methods in de-
tail were described in Table S2.
HPLC method. The 1-phenyl-1,2-ethanediol (PED) and 2-hydroxy-1-
phenylethanone (HAP) were analyzed on C18 chromatographic column
In our previous work, a versatile anti-Prelog carbonyl reductase
AcCR discovered from Acetobacter sp. CCTCC M209061 was char-
acterized, proving that the enzyme was able to catalyze the asymmetric
reduction of various carbonyl substrates with excellent stereo-
selectivity, including acetophenone derivatives and aliphatic ketone
ester. However, the specific activity of AcCR to reduction of ethyl 2-
oxo-4-phenylbutyrate (2a) (1.5 U/mg) and 2-hydroxy-1-phenyletha-
none (3a) (0.4 U/mg), which are useful chiral building blocks, were
(XBridge™ C18, 4.6
φ ×250 mm, 5 μm, Waters Technologies
(Shanghai) Limited) by using HPLC analysis (Agilent 1260 HPLC). UV
detection wavelength of PED and HAP were 215 nm and 245 nm, oven
temperature was 35 °C, and sample size was 20 μL. The mobile phase
was a mixturte of water/acetonitrile (v/v, 3/2, 0.1% trifluoroacetic acid
in water) and the flow rate was 0.5 mL/min. The retention times of PED
(215 nm) and HAP (245 nm) were 6.24 and 7.80 min, respectively. The
2

P. Wei, et al.
MolecularCatalysis479(2019)110613
Fig. 2. Docking of substrates into the active site of WT and mut-AcCR. Figures showed the amino acid residues of enzyme around substrate,a-c: AcCR, mut-G152 L
and mut-E144A docking with 4′-chloro-acetophenone (1a); d-f: AcCR, mut-G152 L and mut-Y189 N docking with ethyl 2-oxo-4-phenylbutyrate (2a); g-i: AcCR, mut-
G152 L and mut-I147 V docking with 2-hydroxy-1-phenylethanone (3a)..
configuration of PED was analyzed on chiral column (OB-H, 0.46 cm φ
×15 cm) by using normal phase HPLC analysis (Agilent 1100 HPLC).
UV detection wavelength of PED was 215 nm, oven temperature was
35 °C, and sample size was 20 μL. The mobile phase was a mixture of n-
hexane/ isopropanol (v/v, 9/1) and the flow rate was 0.7 mL/min. The
retention times of (R)-PED and (S)-PED were 8.51 and 10.21 min.
The calculation methods of the initial reaction rate, the product
yield and the e.e. value were described in our previous report [34].
independent clones) comprising all 20 natural amino acids was initially
constructed by the whole plasmid mutagenic PCR using pGEX-accr as
template. The resulting plasmid (pGEX-mut-accr) was transformed into
BL21(DE3) for the expression of mut-AcCRs.
2.5. Protein expression and purification
The protein expression and purification methods were referred to
our previous reports [33,34].
2.3. Homology modeling and hotspots prediction
2.6. Activity assay
The GenBank accession number of the nucleotide sequence of AcCR
gene is MF419650. Sequence similarity search was performed with the
BLAST program within the Protein Data Bank (PDB). The crystal
structures of 4RF2, 1ZJY, 1NXQ and 1ZK3 in complex with NADH were
used as templates for 3D analysis of the AcCR structure. The MODELER
9.12 program (http://www.salilab.rog) was used to construct the re-
laxed models of AcCR. The model evaluation was conducted using the
PROCHECK program [35] and visualization of AcCR model was pre-
sented by PyMOL (http://www.pymol.org). Hot spots prediction was
performed using the HotSpot Wizard 2.0 server.
Enzyme activity of AcCR and its mutant were determined by mea-
suring the decrease in the absorbance of NADH at 340 nm
(ε = 6.22 mM−1 cm−1
)
using
an
UV–vis
spectrophotometer
(Shinmadzu UV-3010, Japan) at 35 °C for 3 min. The enzymatic reac-
tion catalyzed by AcCR or its mutant was conducted with 0.25 mmol/L
NADH, 20 mmol/L substrate, 5 μL purified enzyme with an appropriate
concentration in 2 mL phosphate buffer (50 mmol/L, pH 6.5). One unit
(U) of enzyme was defined as previous report [34].
2.7. Molecular docking
2.4. Site-saturation mutagenesis
NADH-enzyme docking and substrate-enzyme docking were per-
formed using Molsoft ICM-Pro 3.8-3. All the carbonyl compound sub-
strates were docked into the substrate-binding pocket of AcCR-NADH,
separately. 3D-coordinates (mol2 files) of substrates and NADH were
gained from ZINC database. The molecular docking procedure was
The plasmid of pGEX-accr which harboring AcCR gene from
Acetobacter sp. CCTCC M209061was used for the site-directed muta-
tion. The primers for site-saturation mutagenesis were listed in table S1.
Subsequently, a saturation mutagenesis library (containing about 100
3

P. Wei, et al.
MolecularCatalysis479(2019)110613
Fig. 2. (continued)
executed by using protocol with the default setting. The molecular
docking poses were ranked according to the score functions which were
used to predict their binding affinities and conformations of the mole-
cules at the active sites of enzyme [36,37]. Visualization of molecular
docking results was presented by LigPlot + v.1.4 and Molsoft ICM-Pro
3.8-3.
phosphate buffer (4 mL, 50 mmol/L, pH 6.5) containing 200 mmol/L
substrate, 0.1 mmol/L NADH, and 400 mmol/L isopropanol. The re-
actants were pre-incubated at 35 °C for 10 min, after that a certain
amount of AcCR or its mutant was added to start reaction.
3. Results and discussion
2.8. Asymmetric bioreduction of carbonyl compounds by AcCR and its
variants
3.1. Molecular modeling
The result of homology modeling was shown in Figure S1. As a short
chain dehydrogenase/ reductase, AcCR (Figure S1a) possesses a typical
Rossmann structure, which contained the conserved three-dimensional
Isopropanol was chose to realize the “substrate-coupled” cofactor
regeneration system (Figure S5). The reaction were performed in
4

P. Wei, et al.
MolecularCatalysis479(2019)110613
Fig. 3. Comparison the substrate channel of AcCR and mut-AcCR (Y189 N).
a) the partial view of AcCR (Y189); b) the partial view of mut-AcCR (Y189 N).
Table 2
Kinetic parameters of AcCR and its mutants.
Substrate
Mutant
Km (mM)
kcat (s−1
)
kcat/Km (s−1 mM-1
)
Fold changea
4’-Chloroacetophenone
AcCR
E144A/G 152L
0.3
.02
0.1
0
0.5 × 104
62.83
0.2 × 105
1.3 × 106
1.3 × 104
3.4 × 106
1.0
65.0
1.0
(1a)
0.01
1.5 × 105 148.76
0.2 × 104 54.01
Ethyl 2-Oxo-4-phenylbutyrate
AcCR
G152 L/Y189N
0.12
0.02
261.5
(2a)
0.05
0.2
0.01
0.01
1.7 × 105 88.90
0.6 × 103 21.49
2-Hydroxy-1
-phenylethanone
(3a)
AcCR
I147 V/G152L
2.7 × 103
1.7 × 105
1.0
0.06
0.01
1.0 × 104 65.48
63.0
a
Fold change improvement in kcat/Km over the WT enzyme.
located near the NADH molecule (Figure S2b). There existed hydrogen-
bond interactions between amino acid residues (Ile18, Gly19, Tyr 155
and Ile190) and the pyrophosphate part of NADH, which could increase
the stability of AcCR-NADH interaction [38]. Moreover, it has been
suggested that Ser142 could provide stable interaction to stabilize the
substrate or reaction intermediate through a hydrogen-bond [36].
Tyr155 acted as a general base for initiating the catalysis by transfer-
ring proton to the substrate carbonyl from NADH-C4 [39,40].
Table 3
The relative activity of AcCR mutants in reduction of carbonyl compounds.
Substrate
Enzyme activity [U/mg]a
AcCR mut-
E144A/
mut-
G152 L/
Y189N
mut-
I147 V/
G152L
G152L
4-Chloroacetophenone (1a)
Ethyl 2-oxo-4-phenylbutyrate
(2a)
5.17
1.45
92.70
1.78
2.40
88.96
5.12
2.48
3.2. Exploring key residues of AcCR by hotspot prediction
2-Hydroxy-1-phenylethanone
(3a)
0.37
0.85
1.18
6.44
To improve the catalytic efficiency of the AcCR, mutational hot
spots in the AcCR amino acid sequence were analyzed by using the
HotSpot Wizard2.0 server [41]. Seven hot spot residues (Ala94, Ser96,
Glu144, Met151, Gly152, Ala153, Trp191) were identified (Figure S3a),
and the hot spots were located near the catalytic active sites or the
channel of substrates/products in and out of the AcCR (Figure S3b). In
addition, Ile147 as mutation site was transformed to Val for in-
vestigating the influence of Ile and Val, owing to the contribution of
hydrophobic interactions of Val and Ile on protein surface [42,43].
Moreover, Tyr189 was bulky aromatic amino acid and locate at the
entrance of AcCR active center (Fig. 3), which may hinder the sub-
strates get in and out of the AcCR active center. Therefore, the afore-
mentioned nine residues were identified and altered by site-saturated
mutagenesis (Fig. 1) to obtain mutants of AcCR (mut-AcCR).
4-Fluoroacetophenone (4a)
4-Bromoacetophenone (5a)
3’-Methoxyacetophenone (6a)
4’-Methoxyacetophenone (7a)
Methyl acetoacetate (8a)
Ethyl acetoacetate (9a)
Ethyl-4-chloroacetoacetate
(10a)
4.40
6.05
0.55
0.26
4.19
2.69
6.24
15.06
19.57
1.80
1.41
7.68
2.58
8.43
0.10
1.14
0.24
0.06
3.43
4.83
3.26
2.39
4.92
0.26
0.35
32.58
15.87
18.71
Acetophenone (11a)
0.48
0.35
0.13
1.71
a
0.25 mM NADH, citrate-phosphate bufer (pH 6.5, 50 mM), and 20 mM
subtrate were incubated at 35 °C for 5 min followed by adding a certain amount
of purifed enzyme, the changes in absorbance were recorded in 3 min.
structure and active sites (Ser142-Tyr155-Lys159). The 3D structure of
AcCR almost overlapped with the reference models (Figure S1b).
As shown in Figure S2, the coenzyme binding pocket of AcCR was
broad and the NADH molecule was bounded to AcCR with an extended
conformation at carbonyl ends of the central parallel β-sheet, whose
nicotinamide ring located at the bottom of the lobe region. This or-
ientation was beneficial to deliver NADH-C4 hydride of the nicotina-
mide ring to the carbonyl carbon of the substrate bond in the lobe re-
gion. Moreover, the conserved coenzyme binding motif Gly13-xxx-
Gly17-x-Gly19 and catalytic active sites (Ser142 and Tyr159) were
3.3. Analysis of the activity improvement of most active mutants
The specific activities of the purified mut-AcCRs to three aromatic
ketones (aromatic ketone ester) were determined by measuring the
absorbance decrease of NADH in 2 mL reaction system. The results of
effective mut-AcCRs were summarized in Table 1. As shown in Table 1,
the mut-G152 L was considerably more active with 4.0–11.6 folds im-
proved activity than WT AcCR. For the best substrate (4′-
5

P. Wei, et al.
MolecularCatalysis479(2019)110613
Fig. 4. Asymmetric reduction of prochiral substrates catalyzed by AcCR mutants. Phosphate buffer (pH 6.5, 50 mmol/L, 4 mL) containing 200 mmol/L substrate,
0.1 mmol/L NADH, and 400 mmol/L isopropanol, 35 °C, 200 rpm, 1 mg AcCR mutant.
chloroacetophenone, 1a) of mut-E144A, the specific activity of mut-
E144A was further increased to 47.2 U/mg, which was 9.1 folds higher
than that of the WT AcCR. Meanwhile, for the aromatic ketone ester
substrate (ethyl 2-oxo-4-phenylbutyrate, 2a), the specific activity of
mut-Y189 N was enormously enhanced to 73.4 U/mg, which was 50.6
folds higher than that of WT one. Additionally, with the difficult-to-
reduce ketone catalyzed by WT AcCR, the specific activity of WT AcCR
to 2-hydroxy-1-phenylethanone (3a) was only 0.4 U/mg. Notably, the
specific activity of mut-G152 L to the same substrate could reach to 4.3
U/mg, which was 11.6 folds higher than that of the WT one.
were proposed as the catalytic triad in the carbonyl reductase AcCR.
The carbonyl oxygen atom of substrate formed hydrogen bonds with
Ser142 and Tyr 155 residues, and was protonated from the C4-H atom
of NADH [44,45]. The overlay of the lowest energy docked conforma-
tions of the three substrates into the enzyme active site were shown in
Fig. 2. The most possible conformations of 1a, 2a and 3a in the active
sites of AcCR and mut-G152 L were obtained (Fig. 2a, 2b, 2d, 2e, 2 g,
2 h). As shown in Fig. 2a and b, the distances between the oxygen atoms
of the carbonyl group of 1a and Tyr155 were 2.7 Å and 2.27 Å, re-
spectively. The distance was shortened by 0.43 Å in the two dockings,
which could possibly increase the protonation of oxygen atom of 1a,
resulting in the improvement of the specific activity of mut-G152 L, as
well as the consequence of other two substrates [46]. When the amino
acid residue Glu at site of 144 was mutated to Ala (Fig. 2a and 2c), the
distance in the two dockings between Glu/Ala and oxygen atom of 1a
was shortened by 0.63 Å, which could also increase the protonation,
and the distance between oxygen atom of 1a and amino acid residue
142Ser was shortened by 0.4 Å, which could increase the stability of
enzyme-substrate complex.
3.4. Molecular docking analysis
For the sake of understanding the structural basis of the striking
increase in the specific activity of mut-AcCRs towards the three sub-
strates, the ketones 1a, 2a and 3a were docking into the substrate-
binding pocket of WT AcCR and its mutants, respectively. Such an
analysis would be useful for understanding the increased specific ac-
tivity of mut-AcCRs. Three residues (Ser 142, Tyr 155 and Lys 159)
Compared the result of 2a docking with WT AcCR (4.01 Å), the
6

P. Wei, et al.
MolecularCatalysis479(2019)110613
distances between the oxygen atoms of the carbonyl groups of 2a and
C4-H of nicotinamide mononucleotide (NMN) of cofactor were de-
creased obviously (3.7 and 3.61 Å) (Fig. 2d–f), due to the substrate of 2a
docking with mut-G152 L and mut-Y189 N. The shorter distance sug-
gested a faster hydrogen transfer process which delivered from C4 of
NMN to oxygen atom and improved the specific activity of mut-G152 L
and Y189 N to 2a [47]. Importantly, it was worth noting that the
Tyr189 was bulky aromatic amino acid and located at the channel of
the enzyme active center which could interfere with the larger com-
pounds of 2a and 2b in and out of the active center [48].
As shown in Fig. 3, the replacement of Tyr to Asn at the 189 site
(Asn as a smaller amino acid with similar character of Tyr) could en-
large the channel and gave a rise to 50.58 folds enhancement of the
specific activity, which combined with the distances diminution be-
tween 2a and C4-H of NMN, Tyr155 and Ser142, respectively. The
docking results of WT AcCR, mut-I147 V and mut-G152 L with substrate
of 3a were shown in Fig. 2g–i. From the docking results, it could be
observed that the residues Ser142 forms hydrogen bond with the
oxygen atom of 3a, the distances between the two atoms were 2.68 Å
(WT AcCR), 2.05 Å (mut-G152 L) and 2.32 Å (mut-I147 V), respectively.
The Ser142 as active site in carbonyl reductase was deemed to stabilize
the substrate and reaction intermediate through a hydrogen bond
[36,49]. The decrease of the distances caused the improvement of the
hydrogen-bond interaction between Ser142 and oxygen atom of 3a, and
further increased the stability of substrate-enzyme complex, which
could promote the enhancement of the specific activity of the two mut-
AcCRs towards 3a.
The specific activities of AcCR (mut-G152 L) towards different
substrates (1a, 2a, 3a) were significantly improved after the mutation of
Gly152 to Leu152. Moreover, the specific activity of mut-E144A to 1a,
mut-Y189 N to 2a, mut-I147 V to 3a, mut-W191A to 3a and mut-M151 T
to 3a have been improved to varying degrees from 3.7 to 50.6 folds
(Table 1). Therefore, the combination of mutated site Leu152 and
others were conducted to obtain double-mutants. Finally, Five double-
mutants, mut-E144A/G152 L, mut-G152 L/Y189 N, mut-M151 T/
G152 L, mut-I147 V/G152 L and mut-G152 L/W191A, were achieved.
Among them, the specific activity of the double-mutant mut-E144A/
G152 L to the substrate 1a reached to 92.7 U/mg, which was 17.9 folds
higher than that of WT AcCR (5.2 U/mg), indicating a dramatic im-
provement of the specific activity. For the substrate 2a, the specific
activity of mut-G152 L/Y189 N increased to 88.9 U/mg, which was 61.3
folds higher than that of WT AcCR (1.5 U/mg). Additionally, substrate
of 3a as an important prochiral carbonyl compound has been used to
test the activity of the mut-AcCRs. The specific activity of mut-M151 T/
G152 L, mut-I147 V/G152 L and mut-G152 L/W191A were enhanced by
13.9, 17.4 and 14.5 folds, respectively. Furthermore, the dockings of
the double-mutant mut-AcCRs with different substrates were shown in
Figure S4.
led to a 261-fold improvement in the catalytic efficiency.
3.6. Biocatalytic reduction of various substrates using the AcCR mutants
A series of substrates (1a-11a) with a wide range of structure fea-
tures was applied to characterize the substrate specificity of the mu-
tants. As shown in Table 3, the specific activities of mut-E144A/G152 L
towards 4′-halogenated acetophenones were 15.1, 92.7 and 19.6 U/mg,
respectively, which have improved 3.3–17.9 folds. For 3′- and 4′-
methoxy substituted acetophenone, the specific activity have been en-
hanced 3.3- and 5.4-fold, respectively, which were obviously higher
than that of WT AcCR. The specific activities of mut-I147 V/G152 L
towards β-aliphatic ketone ester and acetophenone have been raised
3.0–7.8 folds. In all the 11 tested substrates, two of the double-mutants
(mut-E144A/G152 L for 1a and mut-G152 L/Y189 N for 2a) have been
improved remarkably with the specific activities more than 88 U/mg.
The asymmetric reductions of various prochiral substrates catalyzed
by the AcCR double-mutants (mut-E144A/G152 L, mut-G152 L/Y189 N
and mut-I147 V/G152 L) have been conducted with 200 mM different
substrates and regeneration of NADH through oxidation of isopropanol
(Figure S6). The results were summarized in Fig. 4. In our previous
study [34], the wild type AcCR could only catalyze the asymmetric
reduction of 50 mmol/L substrates, and the product yields were low
(10.8–66.9%) to most of the investigated substrates except 4′-haloge-
nated acetophenones. The modified mut-AcCRs exhibited outstanding
activity for bioreduction of different substrates with gratifying product
yields ranged from 76.8% to 99.1% as well as excellent e.e. value
(> 99%), which were much more improvement than that of WT one.
The satisfactory results of the bioreduction reaction catalyzed by mut-
AcCRs gave a promising strategy for the synthesis of chiral alcohols in
higher substrate concentrations of various prochiral carbonyl com-
pounds.
4. Conclusions
In conclusion, the specific activity of anti-Prelog carbonyl reductase
AcCR from Acetobacter sp. has been significantly enhanced by site-di-
rected mutations combined with double-mutants. The improvement of
specific activity of the mut-AcCRs is attributed to the enhancement of
hydrogen-bond interaction and the promotion of protonation and hy-
drogen transfer rate. By using various of prochiral carbonyl compounds
at high concentration as substrates, the resultant mut-AcCRs are suc-
cessfully employed for efficient synthesis of enantiomerically pure
chiral alcohols with product yields ranged from 76.8%–99.1% and e.e.
value more than 99%, indicating that the mut-AcCRs are potential for
further catalytic applications.
Declaration of Competing Interest
The shortened distances between the substrates and C4-H of NMN,
as well as the substrates and the catalytic sites Tyr155/ Ser142, en-
hanced the hydrogen-bond interactions in substrate-enzyme complexes,
promoting the increase of the protonation and H-transfer rates to the
oxygen atoms, as well as the stability of the substrate or reaction in-
termediate through a hydrogen-bond [50].
None.
Acknowledgements
The authors gratefully acknowledged the National Natural Science
Foundation of China (No. 21676104 and 21878105), the National Key
Research and Development Program of China (2018YFC1602100,
2018YFC1603400), the Key Research and Development Program of
Guangdong Province (No. 2019B020213001), and the Science and
Technology Program of Guangzhou (201904010360) for partially
funding this work.
3.5. Kinetic studies
The kinetic parameters of the AcCR and its mutants were calculated
by nonlinear regression on the basis of the Michaelis-Menten equation
using OriginPro software. All the kinetic constants of the Wt enzyme
and mutants towards 1a, 2a and 3a were presented in Table 2. Com-
pared with the WT one, all three mutants had lower Km and higher kcat
,
indicating the increase of the catalytic efficiency (kcat/Km). The obvious
decrease in Km confirmed that the binding of variants and substrate
were much stronger than the WT one. Among them, variant G152 L/
Y189 N displayed a decrease in Km and a850-fold increase in kcat, which
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2019.110613.
7

P. Wei, et al.
MolecularCatalysis479(2019)110613
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8