Fine-tuning of the substrate binding mode to enhance the catalytic efficiency of an: Ortho -haloacetophenone-specific carbonyl reductase

Article abstract of DOI:10.1039/c9cy02335f

Carbonyl reductase BaSDR1 has been identified as a potential ortho-haloacetophenone-specific biocatalyst for the synthesis of chiral 1-(2-halophenyl)ethanols due to its excellent stereoselectivity. However, the catalytic efficiency of BaSDR1 is far below the required level for practical applications. Thus, fine-tuning of the substrate binding mode, which aimed at maximum preservation of the positive factors for substrate specificity and stereoselectivity, was proposed as a tentative strategy for enhancing its catalytic efficiency. The designed mutants Q139S, D253Y and Q139S/D253Y showed significantly enhanced catalytic efficiency. Remarkably, the variants Q139S and Q139S/D253Y exhibited a more than 9-fold improvement in catalytic efficiency (k_cat/K_m) toward substrates 6a and 11a, respectively. More importantly, none of the variants caused activity-stereoselectivity trade-off and all variants exhibited excellent stereoselectivity (99% ee). Analysis of variant-substrate complexes showed that the mutations indeed enable the fine-tuning of the substrate binding mode. New strengthening factors for consolidating the productive conformation were introduced while the original positive factors were preserved. Furthermore, at a substrate concentration of 100 mM, recombinant E. coli whole cells expressing the BaSDR1 mutants were successfully applied to the synthesis of several key intermediates of chiral pharmaceuticals, including (S)-1-(2-chlorophenyl)ethanol, (S)-1-(2,4-difluorophenyl)ethanol and (S)-1-(2,6-difluorophenyl)ethanol, with 99% enantiomeric excess, and the conversion reached over 95% in a certain period of time. These results demonstrated the effectiveness of the strategy involving the fine-tuning of the substrate binding mode and the applicability of the designed mutants in efficient reduction of ortho-haloacetophenones.

Full text of DOI:10.1039/c9cy02335f

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DOI: 10.1039/C9CY02335F
ARTICLE
Fine-tuning of the substrate binding mode to enhance the
catalytic efficiency of an ortho-haloacetophenones-specific
carbonyl reductase
Received 00th January 20xx,
Accepted 00th January 20xx
Aipeng Li, a, b Xue Li, b Wei Pang, b Qing Tian, b Ting Wang b and Lianbing Zhang *a, b
DOI: 10.1039/x0xx00000x
Carbonyl reductase BaSDR1 has been identified as a potential ortho-haloacetophenones-specific biocatalyst for the
synthesis of chiral 1-(2-halophenyl)ethanols due to its excellent stereoselectivity. However, the catalytic efficiency of
BaSDR1 is far below the required level for practical applications. Thus, fine-tuning of the substrate binding mode, which
aimed at maximum preservation of the positive factors for substrate specificity and stereoselectivity, was proposed as a
tentative strategy for enhancing its catalytic efficiency. The designed mutants Q139S, D253Y and Q139S/D253Y showed
significantly enhanced catalytic efficiency. Remarkably, the variants Q139S and Q139S/D253Y exhibited more than 9-fold
improvement in catalytic efficiency (kcat/Km) toward substrates 6a and 11a, respectively. More importantly, none of the
variants caused the activity-stereoselectivity trade-off and all variants exhibited excellent stereoselectivity (99% ee).
Analysis of variant-substrate complexes showed that the mutations indeed enable the fine-tuning of substrate binding
mode. New strengthening factors for consolidating the productive conformation were introduced while the original
positive factors were preserved. Furthermore, at a substrate concentration of 100 mM, the recombinant E. coli whole cells
expressing the BaSDR1 mutants were successfully applied to the synthesis of several key intermediates of chiral
pharmaceuticals,
including
(S)-1-(2-chlorophenyl)ethanol,
(S)-1-(2,4-difluorophenyl)ethanol
and
(S)-1-(2,6-
difluorophenyl)ethanol, with 99% enantiomeric excess, and the conversion reached over 95% in a certain period of time.
These results demonstrated the effectiveness of the strategy involving the fine-tuning of substrate binding mode and the
applicability
of
the
designed
mutants
in
efficient
reduction
of
ortho-haloacetophenones.
synthesis of this class of compounds is continuously of great
interest to researchers.
Introduction
It is well know that asymmetric reduction of prochiral ketones
has emerged as a dominant approach for the production of chiral
alcohols in organic synthesis because such process theoretically
converts all substrates to corresponding optically pure
enantiomers.6-8 The conventional chemical processing often suffer
from drawbacks such as environmental toxicity, harsh reaction
conditions, unsatisfactory enantioselectivity, and low yield.9
Biocatalytic asymmetric reduction has been proven to be a green
and preferred alternative for the preparation of chiral alcohols due
to its inherent merits such as environmental compatibility, excellent
enantioselectivity, and mild reaction conditions.10-12 Though
enzyme-catalysed synthesis of one or more chiral 1-(2-
halophenyl)ethanols has been described previously,13-21 the study
specifically focused on the ortho-haloacetophenones-specific
carbonyl reductase was seldom reported. The demand for chiral 1-
(2-halophenyl)ethanols is diverse in the industry, which inevitably
leads to the demand for diverse enzymes. More specific enzymes
will provide more alternative tools for the production of chiral 1-(2-
halophenyl)ethanols. However, it was presumed that the halogen
substitution at ortho-position makes ortho-haloacetophenones
The halogenated aromatic systems were believed to endow
compounds with enhanced permeability and catabolic stability
compared to their non-substituted counterparts.1 As a class of
organic compounds with the chiral carbonic hydroxyl group
adjacent to the halogen atom in the aromatic ring, chiral 1-(2-
halophenyl)ethanols combine the properties of chiral alcohol,
aromatic ring and halogen at the same time. Due to the unique
physical and chemical characteristics, optically active 1-(2-
halophenyl)ethanols and their derivatives have become an
important group of versatile synthons for the synthesis of chiral
agrochemicals, fine chemicals and pharmaceuticals, such as anti-
cancer drug Crizotinib,2 anti-Alzheimer’s drugs,3 nicotinic
4
acetylcholine receptor α7 selective agonist and polo-like kinase 1
inhibitors,5 etc. Thus, development of efficient strategies for the
a. Research & Development Institute in Shenzhen, Northwestern Polytechnical
University, 518057 Shenzhen, China. E-mail: lbzhang@nwpu.edu.cn
b. School of Life Sciences, Northwestern Polytechnical University, 710072 Xi'an,
China.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
inert due to the unique steric and electronic properties of
23
halogen,22,
which impedes the enzymatic performance.
Therefore, there is a requirement for the discovery of enzymes with
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robust capacity or engineering of existing proteins for chiral 1-(2-
halophenyl)ethanols production.
Materials and methods
Materials
View Article Online
DOI: 10.1039/C9CY02335F
Protein engineering provides approaches to create enzymes with
OH
O
25
desired properties.24,
In particular, rational design based on
protein sequences, structures and functional information has been
greatly facilitated by advances in resolution/modelling of the
protein structures and conformational dynamics. It has become an
R
R
NADH NAD+
1b-12b
1a-12a
1 R: H
2 R: 2'-F
3 R: 2'-Cl
4 R: 2'-Br
effective strategy for protein evolution, achieving higher catalytic
9 R: 2'-F-3'-Cl
10 R: 2'-F-4'-Cl
11 R: 2'-F-3'-Br
12 R: 2'-F-4'-Br
5 R: 2'-F-3'-F
6 R: 2'-F-4'-F
7 R: 2'-F-5'-F
8 R: 2'-F-6'-F
28
activity,26 modulating stereoselectivity,27,
expanding substrate
scope,29, 30 and improving enzyme stability.31 It is well recognized
that the amino-acid composition and architecture of substrate-
binding pocket play critical roles in controlling the binding mode of
the substrate, and further impact the catalytic performance of the
Scheme
1
Asymmetric reduction of acetophenone (1a) and ortho-
haloacetophenones (2a-12a) to the corresponding chiral alcohols by BaSDR1.
33
enzyme.32, A substrate binding pocket with ideal properties is
Acetophenone, ortho-haloacetophenones and their corresponding
chiral alcohols of analytical grade were purchased from J&K
Scientific Ltd. (Beijing, China) or Sigma-Aldrich (Shanghai, China).
Plasmid miniprep kit and PCR purification kit were obtained from
Transgen, China. Primers were synthesized by Sangon, China.
PrimerSTAR Max DNA Polymerase and DpnI restriction enzyme
were purchased from TaKaRa (Dalian, China). NADH disodium salt
and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased
from Sangon (Shanghai, China). Ni-NTA column and HisTrap
desalting column were purchased from GE Healthcare Bio-Science,
Sweden. The other chemical reagents used were all of analytical
grade and obtained from local companies.
beneficial for the substrate binding and the product release, while a
pocket with inappropriate properties might cause wrong
positioning of the substrate, leading to the unsatisfactory catalytic
35
performance.34,
Therefore, redesign of the substrate binding
pocket becomes an efficient strategy to tailor enzymes with ideal
32, 33, 36, 37
properties.26,
Accordingly, it was speculated that
modification of the substrate binding pocket based on ortho-
haloacetophenones properties might be an feasible strategy to
achieve efficient enzymes for chiral 1-(2-halophenyl)ethanols
production. However, there is a contradiction between the
maintenance of positive factors which contribute to the
consolidation of the preferred substrate binding mode and the
elimination of the handicaps which hamper the substrate binding.
Though the restructuring of the substrate binding pocket often
could establish new substrate binding modes, the original favorable
conditions are disrupted at the same time, which might have
opposite effects such as loss of substrate specificity and decrease of
stereoselectivity. Therefore, it is a major challenge to balance the
maintenance of the positive factors with the elimination of the
handicaps during a protein engineering process.
Previously, a specific carbonyl reductase (BaSDR1) from Bacillus
aryabhattai was discovered to catalyse the asymmetric reduction of
ortho-haloacetophenones to the corresponding chiral 1-(2-
halophenyl)ethanols with excellent stereoselectivity (Scheme 1).38
This fact suggested that BaSDR1 has a great potential for the
preparation of optically pure 1-(2-halophenyl)ethanols. However,
the catalytic efficiency of BaSDR1 is far from meeting the
requirements of their applications. Therefore, fine-tuning of
substrate binding mode, which aimed at maximum preservation of
the positive factors for substrate specificity and stereoselectivity,
was performed by rational design. The resulting mutants exhibited
Construction of mutants
PCR-based site-directed mutagenesis was performed following the
QuikChangeTM method (Stratagene, La Jolla, CA). The pET30a
plasmid containing the recombinant BaSDR1 gene (GenBank
database under accession number MK374293)38 was used as the
template. The PCR primers are listed in Table S1. The PCR mixture
contained 10 ng of template plasmid, 12.5 μL of 2× PrimerSTAR Max
DNA Polymerase, 0.5 μL of each primer (10 μM) in a total volume of
25 μL. The PCR reaction was carried out under the following
conditions: 98 °C denaturation for 1 min, 20 cycles of 98 °C
denaturation for 10 s, 55 °C annealing for 10 s, and 72 °C extension
for 6 min. The PCR product was purified and then digested with
DpnI at 37 °C for 60 min. The digestion mixture contained 17 μL of
purified PCR product, 2 μL of 10× T Buffer and 1 μL of DpnI. Then
the mixture was transformed into E. coli BL21 (DE3) competent cells
and plated onto Luria-Bertani (LB) agar plates supplemented with
50 μg/mL kanamycin. The entire mutant gene was sequenced to
exclude the introduction of unwanted mutations into the
nucleotide sequence.
significantly enhanced activity towards
a series of ortho-
haloacetophenones. Subsequently, the effects of the altered
substrate binding mode resulted from point mutations on the
enzyme activity of BaSDR1 variants were analysed. And the
effectiveness of design considerations and the mechanism behind
the improved catalytic performance were emphatically discussed.
Finally, whole cell catalyzed synthesis of three optical pure 1-(2-
halophenyl)ethanols, which are applicable to the synthesis of
several compounds for disease treatments, was conducted to
demonstrate the application potential of BaSDR1 variants.
Overexpression and purification of enzymes
All variants were grown in 50 ml LB media supplemented with 50
μg/mL kanamycin at 37 °C with shaking. When the culture reached
an OD600 of 0.6 to 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG,
final concentration 100 μM) was added to induce protein
expression. The cells were subsequently cultured at 30 °C for 6 h
and then harvested by centrifugation. The pellet was washed and
resuspended in 100 mM Na2HPO4-NaH2PO4 buffer (pH 8.0) and then
lysed by sonication. Cell debris was removed by centrifugation at
13800 × g for 15 min, and the supernatant was applied to a Ni-NTA
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affinity chromatography column that had been equilibrated with addition of the substrate. The samples consisted of 5 μM enzyme
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binding buffer (20 mM imidazole, 0.5 M NaCl and 20 mM sodium and 150−3500 μM substrate in potassium DpOhIo: s1p0h.1a0t3e9/bCu9CffYe0r2(313050F
phosphate, pH 7.4). The bound enzyme was eluted with elution mM, pH 7.5). Emission spectra (300−400 nm) were recorded at 280
buffer (0.5 M imidazole, 0.5 M NaCl and 20 mM sodium phosphate, nm
pH 7.4), and then the fractions containing target protein were excitation. The Kd value was determined by the nonlinear least-
desalted with 100 mM Na2HPO4-NaH2PO4 (pH 7.5) by a HisTrap squares method.
desalting column. The expression of the wildtype BaSDR1 as well as
the mutants was examined by SDS-PAGE. Protein concentrations Asymmetric reduction of ortho-haloacetophenones with high
39
were quantified by Bradford method using BSA as the protein substrate loading
standard.
The reaction mixture contained 0.4 g wet resting cells, 100 mM
ortho-haloacetophenone, 5% (w/w) glucose, 1.0 mM Co2+,
Na2HPO4-NaH2PO4 buffer (100 mM, pH 7.5) in a total volume of 10
Enzyme assay and kinetic analysis
Activity of BaSDR1 and the selected mutants was assayed with ml. The biotransformation reactions were carried out at 35 °C with
prochiral ketones as the substrate. One unit of enzyme activity was shaking at 220 rpm. Samples were extracted with ethyl acetate and
defined as the amount of enzyme catalysing the reduction of 1.0 the organic layer was dried over anhydrous Na2SO4. The conversion
μmol of substrate per minute under measurement conditions. The and enantiomeric excess of product (ee) were determined by chiral
assay mixture (1.0 mL) for the reduction of prochiral ketones gas chromatography (GC) analyses. All experiments were conducted
contained 10 mM substrate, 2.0 mM NADH, 5% (w/w) glucose, 1.0 in triplicate if not specified.
mM Co2+ and an appropriate amount of the purified enzyme in 100
mM Na2HPO4-NaH2PO4 buffer (pH 7.5). The reaction mixture was Analytical methods
incubated at 35 °C on a rotary shaker (220 rpm). The reactions were Chiral analysis of prochiral ketones and corresponding chiral
terminated and extracted with 1.0 mL ethyl acetate. The ethyl alcohols was performed on a GC-9790II gas chromatography system
acetate layer was dried over anhydrous Na2SO4 and analysed by gas (Wenling, China) equipped with an FID detector using nitrogen as
chromatography. All experiments were conducted in triplicate if not the carrier gas. The injector and detector temperatures were set at
specified.
250 °C. Prochiral ketones and corresponding chiral alcohols was
The apparent kinetic parameters were determined by measuring determined by chiral capillary column Hydrodex β-TBDAc chiral
the enzyme activity at different substrate concentrations. The column (Macherey-Nagel, Germany, 25 m × 0.25 mm, 0.25 μm film
concentrations from 2.5 to 450 mM were used for determining the thickness). The column temperature program and the retention
kinetic parameters of WT, Q139S and D253Y toward substrate 1a. times were the same as previous report.38
The concentrations from 2.5 to 400 mM were used for determining
the kinetic parameters of WT and D253Y toward substrate 2a. The Computational methods
concentrations from 2.5 to 50 mM were used for determining the The three-dimensional homology model of BaSDR1 was generated
kinetic parameters of WT toward substrate 3a. The concentrations using Modeller in previous report.38 The structure models of
from 2.5 to 100 mM were used for determining the kinetic variants were constructed using Swiss-PDB Viewer. Structural
parameters of D253Y toward substrate 6a. The concentrations from refinement of the models was performed by energy minimization.
2.5 to 50 mM were used for determining the kinetic parameters of Molecular docking was performed using AutoDock Vina 1.1.2.42
WT toward substrate 7a. The concentrations from 2.5 to 50 mM Docking runs were carried out using the standard parameters of the
were used for determining the kinetic parameters of WT and D253Y program for interactive growing and subsequent scoring, except for
toward substrate 8a. The concentrations from 2.5 to 200 mM were the parameters for setting grid box dimensions and center. The
used for determining the kinetic parameters of WT and values of grid box dimensions and centers in docking studies were
Q139S/D253Y toward substrate 9a. The concentrations from 2.5 to set based on the known substrate-binding pocket. Molecular
50 mM were used for determining the kinetic parameters of WT dynamics simulation was conducted as previously described.43 The
toward substrate 11a. The concentrations from 2.5 to 50 mM were conformation of each simulation was analysed by the ptraj program
used for determining the kinetic parameters of WT toward in AMBER package.44 The mmpbsa program in AMBER package was
substrate 12a. For the rest of the enzymes and substrates, the used to evaluate the binding free energy of each complex and the
concentrations from 2.5 to 20 mM were used for determining the decomposed binding free energy of each residues.44
kinetic parameters. The concentration of coenzyme (NADH, 2.0
mM) were kept constant. The constants were calculated from the
Lineweaver-Burk double-reciprocal plot.
Results and discussion
Rational design of BaSDR1
Determination of the dissociation constant Kd
In attempt to upgrade BaSDR1 to a powerful tool for the synthesis
of optically pure 1-(2-halophenyl)ethanols by rational protein
design, the substrate-enzyme complexes were comprehensively
analysed. The complexes of BaSDR1 and two substrates (9a and
10a) were chosen as representative models to illustrate the design
considerations (Fig. 1).
The determination of the dissociation constant Kd was carried out
as described previously with minor modification.40, 41 The affinity of
enzymes to substrates was analyzed with a F97XP fluorescence
spectrophotometer (Leng Guang Tech, Shanghai, China). The
binding of substrates to enzymes was measured by monitoring the
quenching of intrinsic enzyme fluorescence upon incremental
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In the complexes of wildtype BaSDR1 and two substrates (9a and
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DOI: 10.1039/C9CY02335F
10a), the substrate molecules are surrounded by the substrate
binding pocket formed by the residues Q139, V187, 188Q, L196,
Q242, F250, D253 and I291. The substrates showed similar binding
mode in the catalytic cavity. The aromatic ring of the substrates
oriented toward residues D253 and I291, while the carbonyl moiety
Fig. 1 Docking of the substrates 9a (a, b, c) and 10a (d, e, f) into the active site of BaSDR1. The Ser186-Tyr199-Lys203 catalytic triad highlighted by cyan. Blue dash lines,
halogen bonds; Black dash line, hydrophobic interactions. The specific residues are indicated by labels.
located in an absolutely opposite direction, close to Q139 and L196.
Firstly, residues with positive effects for the stabilization of the
The residues V187, 188Q, Q242 and F250 located on the left or right productive conformation were maintained. In the models of the
side of the substrates (Fig. 1). The previous molecular basis analysis enzyme-substrate complex, the preferred substrate binding modes
based on protein-ligand complexes revealed that the steric were facilitated by enzyme-substrate interactions (Fig. 1b and 1e),38
hindrance of halogen atoms and the enzyme-substrate interactions such as the hydrophobic interactions between the aromatic ring of
act in a push-pull manner in regulating the enzyme catalytic substrates and the residues Q188, L196 and V187. In particular, the
performance (Fig. 1, Fig. 2, Fig. 4, Fig. S1, Fig. S2 and Fig. S3).38 Both halogen bonds between the ortho-halogen atoms of substrates and
of them played critical roles in the creation and stabilization of the the proline (P229) oxygen atoms of the peptide backbone were
productive conformation.
main forces for shortening the distance between the key functional
Inspired by these results, strategy involving the fine-tuning of groups, which is an important factor endowing BaSDR1 with ortho-
substrate binding mode was proposed for improving the catalytic haloacetophenones-specificity. Thus, it was speculated that these
activity of BaSDR1. The reason for choosing this strategy is to avoid residues should be preserved due to their positive contributions to
the dramatic changes of the substrate binding mode, which were the substrate binding and the substrate specificity of enzyme.
often reported causing the elimination of the positive factors for Nonetheless, the substitutions of these sites also were performed
substrate binding,28, 45 and further leading to the loss of substrate and further evaluated to test our hypothesis. Secondly, residues
specificity and decrease of stereoselectivity. Therefore, special with negative effects for substrate binding were eliminated or
attention was paid to maximum preservation of the original positive weakened. As depicted in the enzyme-substrate complex models,
factors when the new strengthening factors were introduced for the steric hindrance involving the bulky side chain of Q139 might
achieving the goal of the fine-tuning of substrate binding mode. hamper effective contact of the catalytic residues of the enzyme
With this consideration in mind, the single-mutation variants of with the carbonyl groups of the substrates (Fig. 1). To eliminate or
BaSDR1 were designed as follow.
weaken these negative effects, Q139 was substituted by residues
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with smaller side chains such as alanine, serine, aspartate, leucine trade-off, all variants exhibited excellent stereoselectivity (99% ee)
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and asparagine. Lastly, the new factors with positive effects were toward all substrates tested. Moreover, the DsuObI:s1t0it.u10ti3o9n/Co9fCrYe0s2id3u35eFs
constructed. The docking results indicated that there was extra P229, Q188, L196 and V187 caused adverse effects on BaSDR1
space between the aromatic ring of the substrates and residue activity (Table S2). It is demonstrated that it was a correct choice to
D253, especially for substrates without substitution at para-position keep these residues unchanged.
of the benzene ring (Fig. 1). Moreover, previous analysis indicated
To elucidate the underlying mechanism responsible for the
that the proximity of substrates and the catalytic residues pushed enhancement of activity upon substituting the amino acids, the
by the increased steric hindrance of halogen atoms at para- kinetic parameters of the mutants together with wildtype enzyme
position.38 The residue D253 seems be main contributor to such were determined (Table 2). The kinetic parameters (kcat and Km) of
effect. However, the occurrence of halogen bonds between the the variants for substrates 1a-12a were altered to a varying degree
carboxylic moiety of aspartate (D253) and the para-halogen atoms and all mutants (Q139S, D253Y and Q139S/D253Y) exhibited higher
of the substrates limited the approaching of the substrate carbonyl Table 1 Specific activities of BaSDR1 and its variants
Specific activity(
to the catalytic residue and further prevented the activity of
BaSDR1 (Fig. 1e). Thus, D253 was substituted with residues
harbouring relatively bulky side chains such as glutamate, leucine,
glutamine, phenylalanine and tyrosine to increase the steric
hindrance and eliminate the enzyme-substrate interaction of this
site so as to pushing the substrate closer to the catalytic center and
consolidate the productive conformation. In the cases of residues
Q242, F250 and I291, no obvious interactions were found between
the substrates and these residues. Therefore, mutant involving
those sites were not designed. Accordingly, single-mutation variants
of BaSDR1 were constructed by site-directed mutagenesis and
overexpressed in E. coli BL21(DE3).
Substrate
Enzyme
ee (%)
Yield (%)
U/mg)
WT
0.28±0.11
1.11±0.07
0.73±0.10
99, S
99, S
99, S
97.1±1.1
96.4±0.6
97.6±1.4
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
Q139S/
D253Y
WT
Q139S
D253Y
1a
1.28±0.14
99, S
95.9±2.0
0.34±0.04
1.40±0.13
1.13±0.10
99, S
99, S
99, S
98.4±0.9
97.7±1.6
96.1±2.1
2a
3a
4a
5a
6a
0.74±0.06
99, S
98.1±1.0
0.21±0.04
1.09±0.09
0.43±0.06
99, S
99, S
99, S
95.8±0.8
96.1±1.2
96.7±2.1
0.95±0.06
99, S
97.0±0.7
0.10±0.04
0.22±0.02
0.11±0.03
99, S
99, S
99, S
98.0±1.4
97.9±0.9
98.2±1.7
Asymmetric reduction of prochiral ketones
The asymmetric reduction of substrate 2a was conducted as a
preliminary evaluation of the catalytic performance of the designed
variants using specific activity and stereoselectivity as benchmarks.
(Table S2). All designed variants in residue Q139 (Q139S, Q139A,
Q139D, Q139L and Q139N) exhibited higher specific activities than
the wildtype enzyme. Particularly, variant Q139S increased the
specific activity from 0.34 to 1.40 U/mg, a 4.12-fold enhancement.
Variant D253Y also exhibited enhanced specific activity towards
substrate 2a, with the specific activity value 3.32-fold greater than
that of the wildtype enzyme. The other substitutions at residue
D253 (D253E, D253Q, D253L and D253F) did not lead to better
performance. Based on above results, the catalytic performance of
the variants Q139S and D253Y in the asymmetric reduction of
substrates 1a-12a were further evaluated and compared with that
of wildtype BaSDR1 under the same condition. Indeed, the variants
Q139S and D253Y showed enhanced activity as expected (Table 1).
The enhancement of the variant Q139S activity varies from 1.3- to
8.2-fold toward all substrates tested, while it was 1.1- to 3.9-fold
when the mutant D253Y was utilized as catalyst. These results
inspired us to further investigate how the combination of single-
mutations would affect the enzyme catalytic activity and whether
there would be a synergic effect or not. As shown in Table 1, further
increased activity was observed upon combining these two variants
for several substrates (1a, 7a and 11a). The variant Q139S/D253Y
increased the specific activity from 0.17 to 1.62 U/mg towards 11a,
a 9.5-fold enhancement, which was the highest improvement
among all the substrates tested. Though the variant Q139S/D253Y
exhibited a relatively lower activity toward other substrates
compared with its single mutant counterparts, it has a significant
improvement compared with the wildtype BaSDR1. More
importantly, none of the variants caused activity-stereoselectivity
0.16±0.02
99, S
97.3±1.8
1.10±0.09
3.57±0.21
2.02±0.17
99, S
99, S
99, S
97.7±2.2
98.4±0.6
97.9±1.6
2.86±0.20
99, S
97.9±1.4
0.27±0.06
0.89±0.07
0.40±0.06
99, S
99, S
99, S
96.9±0.7
98.3±1.7
97.5±2.3
0.77±0.11
99, S
96.4±1.9
1.37±0.18
4.69±1.11
2.06±0.36
99, S
99, S
99, S
95.9±2.5
96.3±1.6
97.2±1.4
7a
8a
9a
5.11±1.32
99, S
96.0±0.8
3.76±0.98
4.97±0.15
5.39±0.29
99, S
99, S
99, S
98.3±1.6
98.4±0.4
97.6±0.9
4.81±0.34
99, S
97.3±1.8
0.45±0.09
2.18±0.33
1.54±0.56
99, S
99, S
99, S
98.1±0.9
97.9±1.7
96.9±1.3
1.25±0.48
99, S
97.6±2.4
0.32±0.09
1.14±0.14
0.66±0.11
99, S
99, S
99, S
98.7±0.8
97.5±2.6
98.0±0.5
10a
11a
0.93±0.16
99, S
98.1±1.2
0.17±0.07
1.40±0.10
0.67±0.13
99, S
99, S
99, S
96.3±2.7
97.4±1.4
98.0±0.6
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Journal Name
Q139S/
D253Y
WT
Q139S
D253Y
View Article Online
1.62±0.17
99, S
97.1±0.9
catalytic efficiency (kcat/Km) in comparison wDitOhIt:h1e0.1s0ta3r9t/iCn9gCBYa0S2D33R51F.
Remarkably, the variants Q139S and Q139S/D253Y exhibited
significantly improved catalytic efficiency toward substrates 6a and
11a, respectively, with kcat/Km values up to more than 9-fold greater
0.11±0.06
0.49±0.10
0.28±0.09
99, S
99, S
99, S
97.4±2.3
98.1±0.7
97.6±1.1
12a
Q139S/
D253Y
0.32±0.05
99, S
98.3±2.1
Table 2 Apparent kinetic parameters of BaSDR1 and its mutants
Km
(mM)
kcat
kcat/Km
Km
(mM)
kcat
kcat/Km
Enzyme
Substrate
Substrate
7a
(min−1
)
(min−1 mM−1
0.81±0.11
2.03±0.87
1.84±0.40
)
(min−1
)
(min−1 mM−1
)
WT
45.67±2.31
40.64±1.58
335.48±3.69
36.98±1.26
82.50±2.12
617.28±4.58
30.55±1.67
2.48±0.55
7.99±1.44
136.79±5.32
90.02±6.55
110.88±3.49
4.48±0.99
36.32±2.13
13.87±1.88
Q139S
D253Y
1a
Q139S/
D253Y
6.40±0.87
38.14±1.53
5.96±1.14
1.84±0.24
77.78±1.61
42.31±2.44
WT
301.11±5.33
13.76±1.36
81.62±2.55
409.51±6.21
175.71±3.46
518.29±4.13
1.36±0.78
12.77±1.89
6.35±1.01
24.84±1.68
11.82±1.45
30.85±0.98
172.23±5.09
158.67±3.71
759.64±7.22
6.93±1.11
13.43±1.63
24.62±0.74
Q139S
2a
3a
8a
9a
D253Y
Q139S/
D253Y
10.60±1.53
131.88±3.44
12.44±1.63
4.23±0.13
88.55±1.29
20.94±1.02
WT
25.47±1.21
6.23±1.13
9.35±1.65
18.08±2.31
28.47±1.29
16.98±1.34
0.71±0.03
4.57±1.01
1.82±0.07
141.25±3.68
4.77±0.93
5.82±0.19
450.99±6.66
132.49±2.96
43.68±3.44
3.19±3.14
27.76±1.09
7.51±1.65
Q139S
D253Y
Q139S/
D253Y
8.86±1.39
43.88±2.20
4.95±0.78
19.74±1.28
119.03±1.06
6.03±0.99
WT
11.01±0.69
7.26±1.17
13.21±1.28
7.70±0.69
14.05±1.27
15.48±1.38
0.70±0.04
1.93±0.14
1.17±0.09
3.69±1.10
3.16±0.72
6.02±1.35
8.53±1.11
26.37±2.98
17.72±0.96
2.31±0.63
8.36±1.25
2.94±0.71
Q139S
4a
5a
6a
10a
11a
12a
D253Y
Q139S/
D253Y
6.03±0.65
13.42±0.99
2.23±0.43
2.72±0.64
37.74±1.83
13.89±1.54
WT
16.48±3.21
1.42±0.13
5.36±1.39
75.31±2.22
37.06±1.31
38.38±1.29
4.57±0.22
26.10±1.14
7.16±0.76
34.43±2.33
2.37±0.21
4.45±0.37
64.38±1.22
37.52±1.34
34.70±1.69
1.87±0.87
15.84±2.01
7.80±1.11
Q139S
D253Y
Q139S/
D253Y
2.12±0.11
38.18±2.20
18.01±1.38
1.68±0.09
33.57±1.55
20.02± 1.63
WT
17.93±1.02
9.95±2.01
43.62±4.31
12.78±1.24
65.97±3.47
93.35±2.31
0.71±0.06
6.63±0.65
2.14±0.08
24.66±1.37
3.51±0.56
7.48±1.98
41.68±2.31
33.78±2.68
28.62±1.81
1.69±0.07
9.62±1.23
3.83±0.85
Q139S
D253Y
Q139S/
D253Y
17.05±3.22
73.28±3.79
4.30±0.15
2.87±0.09
27.45±1.67
9.56±1.06
than that of the wildtype. Moreover, the variants Q139S and structural model of the double-mutation variant Q139S/D253Y was
Q139S/D253Y exhibited lower Km values toward all substrates performed. The enzyme-substrate complexes were subjected to
tested. These results indicated that the enhanced enzyme-substrate molecular dynamic (MD) simulation and the average conformations
affinity was the main driving force of the elevated catalytic were extracted. Similar changes were observed in the complex
efficiency of these variants. Specifically, increases in Km values were models of the variant Q139S/D253Y and substrates. The aromatic
observed with a simultaneous increase in kcat values for several ring of the substrates either located in the original orientation or
substrates (1a, 4a, 6a, 8a and 10a) when the mutant D253Y was slightly moved to S186, while the carbonyl moiety was positioned
used as catalyst. Furthermore, specific activity determination (Table closer to S139 and Y199 (Fig. 2, Fig. 4, Fig. S1, Fig. S2 and Fig. S3). In
1) and kinetic analysis (Table 2) both confirmed that there was no addition, the nicotinamide ring of the cofactor NADH were located
synergistic effect of Q139S and D253Y on the enhancement of the at the Re-face of the substrates, a typical Prelog-preferred
catalytic efficiency towards the majority of prochiral ketones. The conformations (Fig. 2, Fig. 4, Fig. S1, Fig. S2 and Fig. S3), leading to
mutant Q139S/D253Y only exhibited higher kcat/Km values towards the corresponding (S)-1-(2-halophenyl)ethanols. This result is in
substrates 1a, 4a, 7a, 10a and 11a compared with Q139S and good agreement with the experimental observations that all
D253Y.
mutations exhibited strict stereoselectivity for (S)-enatiomers in
excellent enantiomeric purity (99% ee) (Table 1).
Most importantly, the mutations effectively executed the design
intent for the majority of substrates, as expected. The introduced
mutations slightly changed the binding modes of the substrates in
the substrate-binding pocket, and the enzyme-substrate
interactions were not obviously influenced for the majority of
enzyme-substrate complexes (Fig. 2, Fig. S1 and Fig. S3). For
Molecular basis of the enhanced activity
In order to confirm the effectiveness of the design considerations
and understand the structural basis leading to such dramatic
enhancement in activity of BaSDR1 toward ortho-
haloacetophenones, docking of a variety of substrates into the
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instance, the halogen bonds between the ortho-halogen atoms of
substrates and the proline oxygen atoms of the peptide backbone
were identified in the complex models of substrate and variant
Q139S/D253Y (Fig. 2, Fig. S1 and Fig. S3). Moreover, the
hydrophobic interactions involving residues Q188 and L196 were
also shown to aid in the positioning of substrates with preferred
conformation (Fig. 2, Fig. S1 and Fig. S3). These results suggested
that the designed mutations could ensure the maintenance of the
positive factors within a certain range of substrates. The strategy
involving the fine-tuning of substrate binding mode is applicable for
improving enzyme catalytic efficiency.
View Article Online
DOI: 10.1039/C9CY02335F
Fig. 3 The distances between the key atoms in the complex models of substrates
and enzymes. Distance Osub-OY199, the distances between the tyrosine hydroxyl
and the oxygen atom of the substrate carbonyl. Distance Csub-C4 NADH, the
distances between the C4 atom of the nicotinamide ring of NADH and the carbon
atom of the substrate carbonyl.
steric flexibility in binding of the substrates and making the
carbonyl moiety of the substrates closer to the catalytic residues.
On the contrary, in the case where D253 was substituted with
tyrosine residue with a relatively bulky side chain, the proximity of
substrates and the catalytic residues pushed by the increased steric
hindrance (Fig. 2B, Fig. 4B, Fig. S1B, Fig. S2B and Fig. S3B). The best
example is given by the dramatic changes of the binding mode of
2',4'-dihaloacetophenones (6a and 10a) compared with other
ortho-haloacetophenones (Fig. S2 and Fig. 4). The close distance
between the para-halogen atoms of the substrates and Y253 makes
them very sensitive to the increase of steric hindrance, and thus the
increase of the steric hindrance at site 253 resulted in the obvious
change of binding mode of those substrates. Previous studies have
repeatedly confirmed that the larger substrate-binding pocket and
lower steric hindrance gives more steric flexibility in substrate
binding and makes substate enter into the active center easier,
resulting to a higher catalytic activity15, 26, 30, 32, 46. Interestingly, it
was also found that the catalytic efficiency of enzyme could been
markedly improved even though the size of the substrate-binding
pocket was decreased14, 24, 33. The variants Q139S and D253Y were
the typical examples of the above two seemingly contradictory
situation, respectively. The mutation of Q139 to a serine residue
decreased the steric hindrance and enlarged the substrate-binding
pocket, while the substitution of D253 with Tyr resulted in
increased steric hindrance and narrowed substrate-binding pocket.
The enhanced catalytic efficiency of those variants indicated that
the access of the substrate to the catalytically active center could
be facilitated by either the increased or decreased steric hindrance.
A pocket matching with substrate structure is beneficial for the
right positioning of the substrate and allows the substrate to gain a
productive binding pose more easily.
Fig. 2 The complex models of substrate 9a and wildtype BaSDR1 (turquoise) or
variant Q139S/D253Y (purple). (a) and (b): Superimposition of wildtype BaSDR1
and variant Q139S/D253Y complex models; (c): The wild type; (d): Q139S/D253Y.
Green dash lines, hydrogen bonds; Blue dash lines, halogen bonds; Black dash
line, hydrophobic interactions. Red dash lines, the distance between the carbonyl
carbon atom of substrate and the C4 atom in the nicotinamide ring of NADH. The
specific residues are indicated by labels.
Obviously, the changes in the position of substrate could be
ascribed to the alterations in the spatial structure of the substrate-
binding pocket. As shown in the models of the enzyme-substrate
complexes, the substrate-binding pocket was reshaped by
substitutions at sites Q139 and D253, which were located at the
opposite ends of catalytic cavity, leading to the substrate binding
mode change and consequently contributed to the enhanced
catalytic efficiency of BaSDR1 (Fig. 2, Fig. 4, Fig. S1, Fig. S2 and Fig.
S3). The mutation of Q139 to a serine residue introduces a smaller
side chain, giving more
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The original halogen bonds were disrupted, and novel halogen
These results were further confirmed by the shortened distances
bonds and hydrophobic interactions were conVsietwruAcrtticelde Onflioner
DOI: 10.1039/C9CY02335F
between the key atoms in the complex models of substrates and
the variant Q139S/D253Y (Fig. 3, Table S3-S8). It was proposed that
the C4 atom of the NADH nicotinamide ring and the hydroxy group
of tyrosine transferred their hydrogen atoms to the carbon atom
and oxygen atom of the substrate carbonyl group in carbonyl
reductase-catalysed reduction, respectively.46, 47 Thus, the distances
between these atoms are the important parameters in determining
the enzyme activity. Compared with wildtype, the distances
between the tyrosine hydroxyl and the oxygen atom of the
substrate carbonyl were shorter (Fig. 3, Table S3-S7), and the closer
distances between the C4 atom of the nicotinamide ring of NADH
and the carbon atom of the substrate carbonyl also were found in
mutant Q139S/D253Y (Fig. 3, Table S8). The hydride transfer was
facilitated by shortening of those distances, and thus, the
engineered enzyme-mediated bioreduction proceeded more
smoothly.
Furthermore, the binding free energy between enzyme and
substrates in the average conformation was calculated by the
mmpbsa program (Table S9). Compared to the wildtype BaSDR1,
the mutant Q139S/D253Y showed a decrease in binding energies
for all substrates tested. The reduced substrate binding energies
could explain the decreased Km and the improved catalytic
efficiency. Moreover, the contributions of residues 139 and 253 to
the binding energy increased, suggesting enhanced interaction
between the substrates and the mutant. These results are in good
agreement with the experimentally determined dissociate constant
Kd. As shown in Table 3, the variant Q139S/D253Y showed much
stabilization of the productive conformation (Fig. 4 and Fig. S2). On
the one hand, it was a reminder that minor differences in substrate
structure exerted great influence on the binding process between
ligand and receptor. The mutants of Lactobacillus kefir short-chain
alcohol
dehydrogenase
even
reduced
substrates
3-
thiacyclopentanone and 3-oxacyclopentanone with obviously
different activities and enantioselectivities, even though these
substrates differ by only the heteroatom (S or O) in the ring.48 Thus,
it is necessary to develop more precise catalytic activity control
strategies for specific substrates. On the other hand, it provided a
persuasive example for the confirmation of the critical role of the
substrate-specific interactions between the enzyme and the
substrate. As shown in Fig. 4, the introduced mutants disrupted the
halogen bonds involving residues P229 and D253, which were main
forces for the generation of the preferred conformations. However,
the predicted decrease in catalytic efficiency was not observed. This
could be ascribed to the construction of new halogen bond
involving residue D242 and hydrophobic interactions involving
residues V187 and Q188. Similar results were obtained in the case
of substrate 6a (Fig. S2). Previous studies also indicated that
enzyme-substrate interactions make great contributions to the
catalytic properties of enzymes.49-51 More notably, it has been
reported that the substitutions of the residues which have no
directed interaction with the substrate could lead to additional
enzyme-substrate interactions formed between other residues and
substrate. These additional interactions result in much stronger
binding affinity.31 The introduction of the substrate-specific
interactions has even been used as a specific strategy for protein
engineering. For instance, the introduction of halogen bond has
been shown as an efficient strategy to optimize the catalytic activity
of a mandelate dehydrogenase.22 The construction of those new
enzyme-
lower
Kd
values
than
that of the wildtype enzyme, suggesting higher affinity of the
Table 3 Dissociation constant Kd and Hill coefficient h of enzymes to the
substrates
Substrate
Enzyme
Kd
h
WT
330.30±5.22
200.34±2.68
428.38±4.36
232.76±3.67
713.37±3.10
523.22±2.31
259.82±2.81
160.77±1.59
523.22±2.71
387.61±1.44
0.86±0.06
0.75±0.08
0.93±0.03
0.91±0.09
0.94±0.05
0.88±0.11
0.82±0.07
0.74±0.12
1.06±0.11
1.00±0.13
5a
Q139S/D253Y
WT
Q139S/D253Y
WT
Q139S/D253Y
WT
Q139S/D253Y
WT
6a
8a
9a
10a
Q139S/D253Y
variant Q139S/D253Y to the tested substrates than the wildtype
enzyme. In addition, the variant Q139S/D253Y showed comparable
Hill coefficient h to those of its parent, indicating that the
mutagenesis did not obviously change the cooperativity of the
enzyme to the tested substrates. Above results indicated that
strategy involving the fine-tuning of substrate binding mode was
efficient to control the enzymatic activity.
Exceptionally, the binding poses of 2',4'-dihaloacetophenones
(10a and 6a) in the variants were totally different from those in the
wild type (Fig. 4 and Fig. S2), even though the enhanced reduction
rates were recorded. In these binding modes, the orientation of the
fluorine atoms at ortho-position were away from P229 and pointed
in completely opposite directions. The protein-ligand interactions
profile also highlighted that the enzyme-substrate interaction
network was significantly changed in the complex models of the
variant Q139S/D253Y and 2',4'-dihaloacetophenones (10a and 6a).
Fig. 4 The complex models of substrate 10a and wildtype BaSDR1 (turquoise) or
variant Q139S/D253Y (purple). (a) and (b): Superimposition of wildtype BaSDR1
and variant Q139S/D253Y complex models; (c): The wild type; (d): Q139S/D253Y.
Green dash lines, hydrogen bonds; Blue dash lines, halogen bonds; Black dash
line, hydrophobic interactions. Red dash lines, the distance between the carbonyl
carbon atom of substrate and the C4 atom in the nicotinamide ring of NADH. The
specific residues are indicated by labels.
substrate interactions allows the substrates to gain a stable binding
pose and thus exhibited higher catalytic activity. Therefore,
enzyme-substrate interactions should be considered as an
important factor in rational design of the enzymes.
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110
In addition, the combination of single-mutations (Q139S and
D253Y) only showed partial synergistic effects on the improvement
of the catalytic activity toward ortho-haloacetophenones (Table 1
and Table S2). Similar phenomenon was previously reported for the
engineered short-chain dehydrogenase/reductase EbSDR8,33 keto
acid reductase LlKAR 14 and amidase Pa-Ami.24 These results further
confirmed that it is necessary to adopt fine-tuning strategy for the
reconstruction of substrate binding pocket in the process of protein
engineering for substrate with unique functional groups. Excessive
modification of the substrate binding pocket might lead to
undesired substrate binding modes and unexpected deterioration
of enzyme catalytic performances. This provides a distinctive
perspective for the generation of suitable mutant proteins with
desired catalytic performance against specific substrates. The
collaborative design strategy for the substitution of the pocket-
forming amino acids should be developed based on the
comprehensive assessment of the properties of the enzyme
catalytic cavity and the substrates.
a
View Article Online
DOI: 10.1039/C9CY02335F
100
90
80
70
60
50
40
30
20
10
0
WT
Q139S/D253Y
O
Cl
3a
0
1
2
3
4
5
6
7
8
9
10 11 12 13
Time (h)
110
100
90
80
70
60
50
40
30
20
10
0
b
WT
Q139S
Asymmetric reduction of ortho-haloacetophenones with high
substrate loading
Enantiopure 1-(2-halophenyl)ethanols are important chiral scaffolds
for the production of pharmaceuticals. For instance, (S)-1-(2-
chlorophenyl)ethanol is a key chiral synthon to access polo-like
kinase 1 inhibitors, a new class of chemotherapeutic agents;5
O
F
F
whereas
(S)-1-(2,4-difluorophenyl)ethanol
and
(S)-1-(2,6-
6a
difluorophenyl)ethanol are applicable to the synthesis of several
compounds in the treatment of sodium channel-mediated
diseases.52, 53 The synthesis of these chiral 1-(2-halophenyl)ethanols
have attracted intense attention from the pharmaceutical
manufacturers. Therefore, the corresponding prochiral ortho-
0
1
2
3
4
5
6
7
8
9
10 11 12 13
Time (h)
110
100
90
80
70
60
50
40
30
20
10
c
haloacetophenones
2'-chloroacetophenone
3a,
2',4'-
difluoroacetophenone 6a and 2',6'-difluoroacetophenone 8a were
selected as the model substrates to test the potential of applying
the BaSDR1 variants. Our previous study indicated that the
recombinant
WT
D253Y
F
O
F
8a
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Time (h)
Fig. 5 Asymmetric reduction of ortho-haloacetophenones 3a (a), 6a (b) and 8a (c)
with high substrate loading by recombinant E. coli whole cells expressing
wildtype BaSDR1 and its variants.
whole-cells overexpressing BaSDR1 could effectively utilize glucose
to recycle the coenzyme and accelerate reaction without the
addition of any exogenous NADH.38 Thus, recombinant whole cells
overexpressing the wildtype BaSDR1 and the mutants were
respectively used to catalyse asymmetric reduction of ortho-
haloacetophenones 3a, 6a and 8a at a concentration of 100 mM on
a 10 mL scale for evaluation of their catalytic performances.
As shown in the conversion profile, all mutants exhibited better
catalytic performances than the wildtype BaSDR1 (Fig. 5). The
conversion of 2'-chloroacetophenone 3a reached over 95% within
5.5 h when the whole cells of Q139S/D253Y were utilized as the
catalyst. In the presence of the whole cells of wildtype BaSDR1, a
lower conversion (49.5%) was achieved within 5.5 h, and the
conversion reached only 70% after 12.5 h (Fig. 5a). In the reduction
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of 100 mM 2',4'-difluoroacetophenone 6a, the mutant Q139S
achieved >95% conversion within 7.0 h, while the starting BaSDR1
afforded only 44.2% conversion within 7.0 h, and 51.2% after 8.5 h
when the reaction became tardy (Fig. 5b). In the case of 2',6'-
difluoroacetophenone 8a by whole cells of D253Y, the substrate
was reduced in 94.8% conversion within 1.0 h and the conversion
reached 98.2% after 1.5 h. In contrast, 41.3% conversion was
obtained after 1 h upon using the whole cells of wildtype BaSDR1,
and the time required to reach 98% conversion was 3.0 h (Fig. 5c).
More importantly, all mutants exhibited high stereoselectivity at a
high substrate concentration, giving corresponding (S)-1-(2-
halophenyl)ethanols in 99% ee. These results suggested that the
BaSDR1 variants could be potential biocatalysts for the asymmetric
reduction of ortho-haloacetophenones.
6
7
8
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Conclusions
In conclusion, the catalytic efficiency of the ortho-
haloacetophenones-specific carbonyl reductase BaSDR1 was
successfully improved by fine-tuning of the substrate binding mode.
The designed mutants exhibited great potential for the synthesis of
various chiral 1-(2-halophenyl)ethanols. The experimental results
showed that the fine-tuning strategy is effective and feasible. It also
could be applied in altering enzyme activity toward other substrates
with special functional groups. This study might help to provide
deepened understanding of the molecular basis for the activity
control of the ortho-haloacetophenones-specific enzymes and
valuable information to guide the future engineering of carbonyl
reductases.
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
The authors greatly acknowledge the financial support from
Science, Technology and Innovation Commission of Shenzhen
Municipality (JCYJ20170815154418207), National Natural Science
Foundation of China (31700700 and 31771577), China Postdoctoral
Science Foundation (2017M623232), The Postdoctoral Research
Project of Shaanxi Province (2018BSHQYXMZZ27), The Natural
Science Basic Research Plan in Shaanxi Province of China
(2018JM3027), The Fundamental Research Funds for the Central
Universities (3102017OQD047 and 3102017OQD049).
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Table of contents entry
Fine-tuning of the substrate binding mode was successfully applied to enhancing the catalytic
efficiency of an ortho-haloacetophenones-specific carbonyl reductase.