Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: Reduction of steric hindrance and change of the stereocontrol element

Article abstract of DOI:10.1039/c9cy02444a

Steric hindrance in the binding pocket of an alcohol dehydrogenase (ADH) has a great impact on its activity and stereoselectivity simultaneously. Due to the subtle structural difference between two bulky phenyl substituents, the asymmetric synthesis of diaryl alcohols by bioreduction of diaryl ketones is often hindered by the low activity and stereoselectivity of ADHs. To engineer an ADH with practical properties and to investigate the molecular mechanism behind the asymmetric biocatalysis of diaryl ketones, we engineered an ADH from Lactobacillus kefiri (LkADH) to asymmetrically catalyse the reduction of 4-chlorodiphenylketones (CPPK), which are not catalysed by the wild type (WT) enzyme. Mutants seq1-seq5 with gradually increased activity and stereoselectivity were obtained through iterative "shrinking mutagenesis." The final mutant seq5 (Y190P/I144V/L199V/E145C/M206F) demonstrated the highest activity and excellent stereoselectivity of >99% ee. Molecular simulation analyses revealed that mutations may enhance the activity by eliminating steric hindrance, inducing a more open binding loop and constructing more noncovalent interactions. The pro-R pose of CPPK with a halogen bond formed a pre-reaction conformation more easily than the pro-S pose, resulting in the high ee of (R)-CPPO in seq5. Moreover, different halogen bonds formed due to the different positions of chlorine substituents, resulting in opposite substrate binding orientation and stereoselectivity. Therefore, the stereoselectivity of seq5 was inverted toward ortho- rather than para-chlorine substituted ketones. These results indicate that the stereocontrol element of LkADH was changed to recognise diaryl ketones after steric hindrance was eliminated. This study provides novel insights into the role of steric hindrance and noncovalent bonds in the determination of the activity and stereoselectivity of enzymes, and presents an approach producing key intermediates of chiral drugs with practical potential.

Full text of DOI:10.1039/c9cy02444a

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DOI: 10.1039/C9CY02444A
Engineering an alcohol dehydrogenase with enhanced activity and
stereoselectivity toward diaryl ketones: reduction of steric
hindrance and change of stereocontrol element
Received 00th January 20xx,
Accepted 00th January 20xx
Kai Wua,b, Zhijun Yanga,b, Xiangguo Menga,b, Rong chena, Jiankun Huanga and Lei Shao*b,c
DOI: 10.1039/x0xx00000x
Steric hindrance in the binding pocket of alcohol dehydrogenase (ADH) has a great impact on activity and stereoselectivity
simultaneously. Due to the subtle structural difference between two bulky phenyl substituents, the asymmetric synthesis of
diaryl alcohols by bioreduction of diaryl ketone is often hindered by low activity and stereoselectivity of ADH. To engineer
ADH with practical properties and investigate the molecular mechanism behind asymmetric biocatalysis of diaryl ketones,
we engineered ADH from Lactobacillus kefir (LkADH) to asymmetrically catalyse the reduction of 4-chlorodiphenylketones
(CPPK), which are not catalysed by the wild type (WT) enzyme. Mutants seq1-seq5 with gradually increased activity and
stereoselectivity were obtained through iterative “shrinking mutagenesis.” The final mutant seq5
(Y190P/I144V/L199V/E145C/M206F) demonstrated the highest activity and excellent stereoselectivity of >99% ee.
Molecular simulation analyses revealed that mutations may enhance activity by eliminating steric hindrance, inducing a
more open binding loop and constructing more noncovalent interactions. Pro-R pose of CPPK with a halogen bond formed
pre-reaction conformation easier than pro-S pose, resulting in high ee of (R)-CPPO in seq5. Moreover, different halogen
bonds formed due to different positions of chlorine substituents, resulting in opposite substrate binding orientation and
stereoselectivity. Therefore, the stereoselectivity of seq5 was inverted toward ortho- rather than para-chlorine substituted
ketones. These results indicate that the stereocontrol element of LkADH was changed to recognise diaryl ketones after steric
hindrance was eliminated. This study provides novel insights into the role of steric hindrance and noncovalent bonds in the
determination of activity and stereoselectivity of enzymes, and presents an approach producing key intermediates of chiral
drug with practical potential.
cloperastine, which can be prepared through the symmetric
reduction of diaryl ketone CPPK. Levocloperastine is faster
acting and causes greater reductions in the intensity and
Introduction
Alcohol dehydrogenases (ADHs, EC 1.1.1.1) constitute a large frequency of coughing in recipients compared with racemic
family of NAD(P)(H)-dependent oxidoreductases. Multiple cloperastine7. The chemical synthesis of chiral diaryl alcohols
ADHs efficiently catalyse asymmetric bioreduction for chiral often involves expensive Ru catalysts or resolving agents8.
pharmaceutical intermediate production with great industrial Therefore, the alternative strategy for production is bio-
potential1. Biocatalyst activity and stereoselectivity are two key reduction by ADH, which is considered the more ecologically
properties. Based on previous studies involving stereocontrol and economically viable route for synthesis.
mechanisms2-3, the stereoselectivity of ADH originating from Due to the important role of steric hindrance in chiral
the preference of specific binding orientation of prochiral recognition and activity, from the view of rational design, it can
ketones and steric hindrance in binding pockets plays an be inferred that the elimination of steric hindrance in the
important role in determining the binding orientation of binding pocket has a double effect of providing improved
substrates. The prochiral ketones bearing substituents differ in ligand-protein interactions with the enhancement of activity
size and bind to small and large binding pockets of ADH. toward bulky CPPK, and potentially a low stereoselectivity,
Therefore, the ADHs generally demonstrate high activity and unless a different stereocontrol element takes over. In this
stereoselectivity toward bulky-small ketones. However, when study, we aimed to create an ADH variant with enhanced
ADH catalyses diaryl ketones bearing two bulky substituents activity and stereoselectivity toward CPPK without property
with similar phenyl groups, low activity and stereoselectivity are trade-offs. The molecular basis of ADH from Lactobacillus kefiri
often observed4-5
.
DSM 20587 (LkADH), which has a resolved crystal structure2,
Diaryl ketones can be asymmetrically reduced to the was selected for engineering. Naturally occurring LkADH shows
corresponding chiral alcohol. Optical pure diaryl alcohols are a preference for the reduction of bulky-small ketones as
important pharmaceutical intermediates6. Among these opposed to bulky-bulky ketones. It is difficult for the phenyl
alcohols, (R)-(4-chlorophenyl)(phenyl)methanol (CPPO) is the group of CPPK to bind to a small pocket, which results in an
critical intermediate in the production of laevorotatory inactive binding conformation. Reduction using LkADH wild-
type (WT)-only produced a conversion of less than 0.1%. This
result further suggests that steric hindrance prevents optimal
a. School of Pharmacy, Shanghai University of Medicine & Health sciences, 279
substrate interactions. Moreover, the subtle differences
between the two phenyl groups of CPPK results in difficulty in
chiral recognition for LkADH. In this study, we constructed
focused libraries consisting of limited amino acids with sites
Zhouzhu Highway, Pudong New Area, Shanghai 201318, China.
b.. Microbial Pharmacology Laboratory, Shanghai University of Medicine & Health
sciences, 279 Zhouzhu Highway, Pudong New Area, Shanghai 201318, China.
c. State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai
Institute of Pharmaceutical Industry, 285 Gebaini Rd., Shanghai 200040, China.
*E-mail address: shaolei00@gmail.com (L. Shao).
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selected in or around the catalytic pocket to screen variants adding 1 mL isopropanol and incubating the mixtuVrieewaAtrt6ic5le°OCnflioner
DOI: 10.1039/C9CY02444A
with enhanced activity and stereoselectivity. A 5-point mutant 10 min. The samples were analysed after filtering by HPLC to
(seq5) with reduced steric hindrance and dramatically increased determine the conversion ratio and ee.
properties was obtained. The molecular docking and dynamic Purification of LkADH and mutants
simulations of the WT and mutants further revealed the source
of enhanced activity and stereoselectivity. Moreover, huge For purifying WT and seq1-5, the respective genes flanked by
differences in chiral recognition between the WT and seq5 were NdeI and HindIII sites were expressed in pET-28a with an N-
discovered, which indicated that the stereocontrol element can terminal His-tag. Resting cells expressing mutant LkADH were
be converted in engineered ADH.
Experimental Section
Materials
disrupted by sonication, and cell debris/inclusion bodies were
removed by centrifugation at 10,000 × g at 4°C for 15 min.
Soluble cell-free extract was loaded onto a nickel column that
was pre-equilibrated with 50 mM Tris-HCl (pH 7.6) binding
buffer. The bound recombinant enzyme was eluted using a
binding buffer containing increasing concentrations of
Ketones, racemic sec-alcohol standards, and NADPH used in this imidazole (20–200 mM), according to a standard protocol. The
study are commercially available. The conversion ratio and purified enzyme was obtained using 200 mM imidazole. The
enantiomeric excess after bioreduction was analysed using purified enzyme was further used to determine the specific
chiral high-performance liquid chromatography (HPLC; Chiralcel activity and stereoselectivity.
OB-H/AD-H/OD-H column [250 × 4.6 mm]). The KOD-Plus-neo
DNA Polymerase was obtained from TOYOBO CO., Ltd. Measurement of activity and ee of WT and seq5
Restriction enzyme Dpn I was procured from Thermo Scientific.
Oligonucleotide synthesis and DNA sequencing were conducted Specific activities of WT and seq5 were spectrophotometrically
by GENEWIZ. The TIANprep Mini Plasmid Kit used for plasmid assayed by measuring the change in NADPH absorbance at 340
extraction was procured from Tiangen Biotech.
nm in 1 min. The reaction system contained the appropriate
weights of the purified enzymes, 0.625 mM NADPH, 5 mM
substrates, and 100 mM Tris-HCl (pH 7.6) buffer, at a final
reaction volume of 200 μl. The kinetic parameters of WT and
Mutant construction, expression, and screening
Active pocket iterative mutagenesis is based on information seq5 to CPPK were determined by increasing the substrate
regarding the protein active pocket structure, with iterative concentration from 0.3 to 10 mM at 30°C. Initial velocities at
cycles of mutagenesis performed at rationally chosen sites using different substrate concentrations were used to generate a
smaller glycine, alanine, cysteine, serine, proline, and valine Lineweaver−Burk plot (1/v vs. 1/[S]). The product ee of the
residues to replace the original residues. The full-length LkADH corresponding 1a-9a was determined by chiral HPLC. The chiral
gene was expressed using pET-21b, and the recombinant analysis methods and retention time of R and S enantiomer are
plasmid was used as the PCR template for “shrinking listed in Table S3. The absolute configuration was determined
mutagenesis.” The obtained PCR products were digested using by comparing the retention time with previous reports.
DpnI to remove parent plasmids. The digested PCR products
were transformed into Escherichia coli DH5α cells. Plasmids Molecular docking and MD simulations
containing the mutant gene were then extracted and
retransformed into E. coli BL21 (DE3) cells for mutant enzyme The crystal structure of LkADH was obtained from the protein
expression.
data bank (PDB ID 4RF2)2. The virtual mutation was constructed
The gene encoding LkADH (accession number: AY267012) from using Discovery Studio 4.0. The substrate docking was
Lactobacillus kefiri DSM 20587 was expressed using pET-21b. A performed using the Autodock 4.2 program. CPPK, 2-chloro-1-
single transformant was cultured at 37°C for 12 h and was then phenylethanone, and 4-chloro-1-phenylethanone were docked
transferred to 100 mL fresh Luria–Bertani (LB) medium and into the substrate-binding pocket of the LkADH-NADPH
cultured at 37°C. The culture was induced by adding 0.1 mM complex by flexible dock. 3D-coordinates (mol2 files) of
isopropyl β-D-1-thiogalactopyranoside when its optical density substrates were downloaded from the ZINC database.
at 600 nm reached 0.6. After induction at 20°C for 20 h, resting AutoDock tools (ADT, version 1.5.6) were used for enzyme and
cells were harvested by centrifuging at 8500 × g for 10 min and substrate preparation. To encompass the entire substrate-
were then resuspended in 100 mM Tris-HCl (pH 7.6) with a cell binding pocket, the docking box was set to a size of 50 × 50 × 50
concentration of 80 mg·mL-1.
grid points with a grid spacing of 0.375 Å. The box centre was
Resting cells expressing mutants were used as biocatalysts in set as X=21.557, Y=-29.525, and Z=26.307. To obtain a
the reaction of CPPK to determine the conversion and ee. The reasonable initial conformation for MD, residues S143, Y156,
screening reaction mixture (500 µL) included 200 µL of V144, C145, L195, V196, V199, F206, and Y249 of seq5 were set
suspended resting cells expressing mutants, 25 µL of 5 mM as flexible. The obtained conformations, in which the carbonyl
NADP stock, and 50 µL of 200 mM CPPK dissolved in oxygen had hydrogen bond interactions with Tyr156 and the
isopropanol. The reaction mixture was incubated at 30°C with distance between carbonyl carbon and NADPH-C4 atoms was
rotation at 200 rpm for 30 min. The reaction was terminated by less than 4.5 Å, were considered reasonable. The reasonable
2 | J. Name., 2012, 00, 1-3
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conformation with the lowest binding energy was selected for free energies of CPPK in seq5 and seq1 were calVciuewlaAtreticdle fOrnolimne
DOI: 10.1039/C9CY02444A
further analysis. The ligand interaction was analysed using the 1000 frames extracted from ten independent 4 ns simulations
receptor-ligand interactions tool of Discovery Studio V4.0 with stable root-mean-square deviation, using the Generalised
(Biovia, USA).
Born surface area method implemented in MMPBSA.py script
MD simulation was performed using the Particle Mesh Ewald of Amber 1813.
Molecular Dynamics module implemented in the Amber 18
suite9, with the ff14SB force field used for the protein system, Gram-scale preparation of (R)-CPPO and (S)-CPMO
and the GAFF force field used for the ligands10. The NADPH
parameters were obtained from the AMBER parameter We scaled-up the production (200 mL) of (R)-CPPO and (S)-
database11. The ANTECHAMBER module and Gaussian 09 were CPMO to determine the practical application of the engineered
used to calculate the CPPK RESP atom charges. Hydrogen atoms enzyme seq5. The resting cells expressing seq5 were used for
and sodium ions (to neutralise the negative charges) were the scale-up preparation. Recombinant glucose dehydrogenase
added to the protein using the tleap utility. Each simulation (GDH) from Bacillus subtilis CGMCC 1.1398 and glucose was
system was immersed in a cube of TIP3P explicit water, used for NADPH recycling. The pH was automatically adjusted
extending to 12 Å outside the protein on all sides. Water to 7.6 by titration with 2.0 M Na2CO3 solution. The bioreduction
molecules were treated using the SHAKE algorithm, and the of CPPK was initiated at 30°C by adding 17.3 g of CPPK dissolved
long-range electrostatic effects were considered using the in 60 mL methyl tert-butyl ether to 140 mL of 100 mM Tris-HCl
particle mesh Ewald method. The binary complex of protein- (pH 7.6), which contained 0.25 mM NADP, 17.5 g glucose, 16 g
NADPH without substrate being bound, the ternary complex resting cells expressing seq5, and 3.2 g resting cells expressing
obtained from the molecular docking of seq1-NADPH-CPPK, and GDH. The bioreduction of CPMO was initiated at 30°C by adding
complexes with pro-R/S docking poses of the seq5-NADPH- 17.4 g of 5a dissolved in 20 mL methanol to 180 mL of 100 mM
CPPK were treated as follows. The water molecules and ions Tris-HCl (pH 7.6), which contained 0.25 mM NADP, 16.4 g
were relaxed to minimise the energy during the 10,000 glucose, 10 g resting cells expressing seq5, and 3.2 g resting cells
minimisation steps with the protein and ligands restrained. The expressing GDH. The conversion ratio was monitored using
backbone of the protein was restrained with the other section HPLC and the reaction mixture was extracted by ethyl acetate
relaxed to minimise the energy. The whole system was then after reaction completion.
minimised without the restraints during the 10,000
minimisation steps. After energy minimisation, the system was Results and discussion
gradually heated in the NVT ensemble from 0 to 303 K over 200
ps. This procedure was followed by 200 ps of NPT simulation at Construction and screening of the mutagenesis library
303 K and 1 atm pressure using the Langevin dynamics
algorithm with the complex constrained. Equilibration of 200 ps CPPK is a typical bulky-bulky ketone, which is flanked by two
was performed with the complex constrained. All the positional phenyl rings. The two bulky phenyls may prevent binding near
constraints in energy minimisation and equilibration used a catalytic residues. The catalysis of LkADH involves a highly
force constant of 2.0 kcal mol−1 Å−2. Three independent conserved triad consisting of Ser143-Tyr156-Lys1602. Ser143
productive simulations of WT, seq1, and seq5 were performed stabilises the substrate. Tyr156 acts as a general acid/base
without any restraints for 200 ns. A time-step of 2.0 fs was used, initiating catalysis by transferring its proton to the carbonyl
and coordinates of the system were saved every 2 ps during group of the substrate. NADPH transfers hydride to carbonyl
production. The pocket volumes were calculated using the carbon atoms. Therefore, the distance between the substrate
POVME2 program12. The WT, seq1, and seq5 used the same carbonyl oxygen and Tyr156-OH and the distance between the
parameters to describe the binding pocket in POVME 2.0. Based carbonyl carbon and NADPH-C4 are critical for catalysis. A rigid
on the algorithm of this program, the Grid Spacing was set as docking experiment was designed to explore the binding
0.5 for better accuracy. The centre of Points Inclusion Sphere conformation of this inactive substrate. Large distances
was set as X=45, Y=42, Z=27 and the radius was 10 Å. The centre (distance OHTyr156-O1CPPK =12.1 Å and C4NADPH-C1CPPK =13.4 Å,
of Points Exclusion Sphere was set as X=50, Y=44, Z=38 and the respectively) were observed in docking conformation,
radius was 6 Å. The default Distance Cutoff value was used. The indicating steric hindrance by the side chains within the binding
Convex-Hull-Clipping option was set as “True” to increase pocket preventing CPPK access and catalysis. Based on this
accuracy. The centre of Contiguous Pocket Seed Sphere was set analysis, the primary objective of our engineering project was
as X=45, Y=42, Z=26 and the radius was 7 Å. The Contiguous to create a binding pocket permissive for CPPK to facilitate
Points Criteria was set as 3.
catalysis.
To calculate the proportion of pre-reaction conformations of Based on the structural information of the binding pocket and
the pro-S and pro-R pose, each pose obtained from the docking catalytic mechanism, we selected five residues around the
result underwent 4 ns of MD simulation with ten replicas. catalytic triad as the target for “shrinking mutagenesis” with an
Distances were calculated with the cpptraj module. The plot of iterative strategy aimed at the expansion of the original small
probability density distribution of the distances of C4NADPH
-
binding pocket (Figure S1). Based on the CPPK hydrophobicity
C1CPPK and OHTyr156-O1CPPK was calculated using 2D Kernel (logP=3.57), van der Waals volumes, and charges of residues,
Density estimation provided by OriginPro 2018. The binding the
bulkier
residues
with
large
volumes
This journal is © The Royal Society of Chemistry 20xx
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(Tyr190/Ile144/Leu199/Glu145/Leu195) were selected for in the smallest VDW volume. However, it has also lost the
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mutagenesis to aliphatic residues (Ala/Pro/Val/Gly) and polar necessary hydrophobic interactions DfOoI:r10.1C0P3P9K/C9CbYi0n2d4i4n4gA.
residues (Ser/Cys) with smaller volumes. Basic and acidic amino Compared with Ala, Val demonstrated enhanced activity and
acids were excluded because of their hydrophilicity. The van der stereoselectivity. This may be because of its relatively large
Waals volumes of 20 residues are listed in Table S1. Based on hydrophobic isopropyl group providing better hydrophobic
the crystal structure of LkADH, Leu199, Glu145, and Leu195 interactions. In a previous study, Val was also used for
were in different α-helixes, and the Tyr190 was located in a loop manipulating stereoselectivity in epoxide hydrolase to achieve
region. Based on previous reports, Pro is the worst helix former much better results than using Ala17. The stereoselectivity of
due to its lack of an amide hydrogen for main chain hydrogen seq2 further increased from 72.1% to 89% ee.
bonding and because of its unique geometry14. Mutations to Pro Seq2 was then used as a template to mutate Leu199 obtaining
at sites Leu199, Glu145, and Leu195 could destabilize the α- seq3 (Y190P/Y144V/L199V). The conversion of seq3 increased
helix structure and result in inactive variants. However, proline to 31.3%, and increased ee to 94.7%. Using seq3 as a template,
is frequently found in turn and loop structures of proteins15. Glu145 was selected for mutagenesis, creating seq4
Therefore, only the site in loop region (Tyr190) was mutated (Y190P/I144V/L199V/E145C). The stereoselectivity of seq4 was
into Pro, and sites in α-helixes (Leu199, Glu145, and Leu195) more than 99% ee, and the conversion ratio further increased
were excluded.
to 33.9%. Using seq4 as a template, all six mutations
The iterative mutagenesis is a powerful approach for semi- demonstrated decreased activity at Leu195, indicating that
rational design16. With structural information on the binding leucine has a favourable interaction with CPPK.
pocket, the sequence space could be reduced by selecting To increase the activity of seq4 further, a flexible docking
limited residues in or near the binding pocket. Beneficial analysis was performed. Based on the docking results of seq4
mutations with additive and synergistic effects could be (template used for M206F), the CPPK in the pre-reaction
accumulated using several rounds of mutagenesis.
conformation was found to interact with Met206 and Tyr249.
Tyr190 was the residue with the largest volume (141 Å3) in the Besides, as the CPPK is a hydrophobic compound with two
binding pocket. Therefore, it may be the key residue preventing aromatic rings, this indicates that increasing hydrophobic effect
the binding of the large groups in the small binding pocket. This and constructing π-π interaction will probably help to stabilize
site was targeted first as changes to this site may result in higher pre-reaction conformation. Among the amino acids, only Phe,
activity. Using resting cells expressing mutants, all six mutants Tyr, and Trp have an aromatic ring that allows them to form π-
at sites Y190 produced measurable CPPO. Among them, Y190G, π interaction with CPPK. Moreover, Phe has the smallest van der
Y190A, and Y190C demonstrated a conversion ratio of more Waals volume among the aromatic amino acids. Therefore, it
than 30%, a dramatic increase when compared with WT. was chosen for the site-directed mutation of M206F to
However, these mutants demonstrated low ee as 3%, 30.5% and construct π-π ‘T’-shaped interaction and enhance the
31.3%, respectively. The Y190P (seq1) mutation demonstrated hydrophobic effect. After virtual mutation, docking results
the highest ee of 72.1% with a medium conversion ratio of demonstrated that the phenyl ring of CPPK interacted with
20.1%. Based on this, we used Y190P as the template to perform Tyr249 and Phe 206 by π-π ‘T’-shaped interaction and formed a
the second cycle of mutagenesis at Ile144. The location of Ile144 densely packed hydrophobic region. In addition, based on a
was quite close to catalytic residue Ser143, which may be previous report, the π-π interactions had been successfully
sensitive to residue change. Therefore, only similar small designed to affect transition-state binding for catalysis18.
aliphatic mutations of Val, Ala, and Gly were constructed. Therefore, from the view of rational design, a site direct
Y190P/I144V (seq2) demonstrated an increased conversion mutation of M206F was performed. As expected, seq5
ratio of 26.1%, and was the only active mutant at site I144. The (Y190P/I144V/L199V/E145C/M206F) further increased the
I144G and I144A lost activity toward CPPK. Gly is an amino acid conversion to 46% and maintained an ee value of more than
that has a single hydrogen atom as its side chain, which results 99%.
Table 1. Results of activity and stereoselectivity screen.
Template
WT
Mutation Conversion ratio (%)
ee (%)
Configuration
none
Y190G
Y190A
Y190S
Y190C
Y190P
Y190V
I144G
<0.1
31
−
3
−
R
R
R
R
R
R
−
35
30.5
−
0.9
WT
24.3
20.1
4.32
<0.1
31.3
72.1
43
Seq1:
−
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Y190P
I144A
I144V
L199A
L199S
L199C
L199V
L199G
E145G
E145A
E145S
E145C
E145V
L195G
L195A
L195S
L195C
L195V
<0.1
26.1
4.2
−
−
R
R
R
R
R
−
R
R
R
R
−
R
−
R
−
−
View Article Online
DOI: 10.1039/C9CY02444A
89
90
4.5
>99
98.5
94.7
−
Seq2:
Y190P/I144
V
12.3
31.3
<0.1
2.2
>99
95
27
Seq3:
Y190P/I144
V/L199V
5.3
85.9
>99
−
33.9
<0.1
8.9
>99
−
Seq4:Y190
P/I144V/L1
99V/E145C
<0.1
7.7
>99
−
<0.1
<0.1
−
Seq4:Y190
P/I144V/L1
99V/E145C
M206F
46
>99
R
The conversion ratio and enantiomer excess of all mutants activity and stereoselectivity. The final mutant seq5
created using iterative mutagenesis are listed in Table 1. demonstrated a kcat of 246 times higher than seq1. The kinetic
Through five rounds of mutagenesis with minimal screening, we characterisation of the variants indicated that the enhancement
obtained
a
five-point
mutant
with
seq5 in catalytic efficiency was predominantly the result of an
dramatic improvement in kcat. Based on the Michaelis-Menten kinetics, it
was reasonable that a lower Km was obtained in seq1 than seq5
because the rate of reaction step of seq1 is extremely slow
relative to the rate of substrate binding. Saturation will thus be
reached at fairly low substrate concentrations. On the contrary,
(Y190P/I144V/L199V/E145C/M206F)
enhancement of activity and stereoselectivity.
a
Investigation of the source of activity toward CPPK
To further confirm the gradual enhancement of catalytic as the reaction step of seq5 is relatively fast, it will take a large
efficiency, the mutants seq1-5 were purified to allow the quantity of substrate to reach saturation. An engineered
determination of the kinetic parameters (Figure S2). The epoxide hydrolase with active tunnel expanded towards the
parameters are listed in table 2. The mutation of seq1 produced bulky α-naphthyl glycidyl ether also showed an increased Km
low but detectable activity compared to WT, indicating that the value and enhanced kcat compared with WT19, which was similar
reduction of hindrance at Tyr190 was critical for increased to our findings.
activity. Seq1 paved the way for further enhancements in
Table 2. Kinetic parameters of variants seq1-5
Mutant
Km (mM)
Vmax (µmol·min-1·mg-1)
kcat (min-1)
Seq1 (Y190P)
Seq2 (Y190P/I144V)
Seq3 (Y190P/I144V/L199V)
Seq4 (Y190P/I144V/L199V/E145C)
Seq5 (Y190P/I144V/L199V/E145C/M206F)
0.54
1.08
1.13
3.30
3.14
0.0055
0.0426
0.0710
0.1316
1.2811
0.15
1.2
2.0
3.8
36.9
The enhanced activity implied that hindrance in the binding WT and seq5 were calculated as 163.25 Å3 and 203.63 Å3,
pocket of the WT enzyme was reduced, and that the pocket respectively. The engineered seq5 reduced the steric hindrance
expanded to accept the bulkier CPPK substrate. To understand and achieved an expanded binding pocket, as expected.
the differences in the enzymatic activity, the pocket volumes of
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reason for the dramatically increased pocket volume in seq1
View Article Online
DOI: 10.1039/C9CY02444A
and seq5.
The one amino acid substitution Y190P of seq1 with
dramatically increased volume suggests that proline was
responsible for this prolonged open state of the binding pocket.
Proline is often found in the secondary structure of the β-turn
and is often used to induce a turn in the loop20-21. This might be
because the cyclical structure of proline gives rotation. The
rotation could bend the binding loop with a greater probability
of opening rather than closing, which further resulted in large
pocket volume. The different proportions of the closed or
opened state of the binding pocket could be manipulated by
different mutations. Contrary to mutation Y190P, the previously
reported mutation A94F promotes a slightly more closed
binding loop because Phe94 makes van der Waals contact with
Leu1952. The mobile loop regions located at the entrance of the
active site pocket are often considered as the gates that control
the entry and exit of substrates and products, in addition to
shielding the active site from the bulk solvent during catalysis22-
23. Therefore, the results above indicate that reduced steric
hindrance and a wider entrance for substrate access was
obtained in seq1 and seq5.
Figure 1. Curves of binding pocket volumes and overlays of representative
snapshots for the substrate binding loop. A. Differences in pocket volumes of WT,
seq1, and seq5 during simulations. B. Overlay of the representative structures of
the binding loop of WT, seq1, and seq5. WT, seq1, and seq5 are coloured grey,
red, and blue, respectively.
Figure 2. Docking conformation and interactions of CPPK in seq5. A. Binding mode of
CPPK in seq5. The hydrogen bonds are shown in green; π-sulphur interactions in
orange; π-π interactions are in purple; the π-alkyl interaction in light pink; and the
halogen bond in blue. B. Binding energy decomposition of key residues in the pocket.
Considering the dynamic motion of the enzyme-NADPH
complex, we further calculated the pocket volumes of WT, seq1,
and seq5 during three independent MD simulations of 200 ns
(in total 600 ns). As illustrated in Figure 1A, significant
differences in volume distribution were found. Pocket volume
distribution analysis revealed that seq1 and seq5 exhibit larger
average pocket volumes as well as broader distributions. The
WT exhibits a smaller average pocket volume and a narrower
distribution. However, this difference cannot be solely
explained by the replacement of bulky residues with smaller
ones. Furthermore, the representative structures of WT, seq1,
and seq5 during simulation were overlaid and compared. The
comparison of the secondary structure revealed that the
substrate-binding loop (residues 190-210) was much more open
in seq1 and seq5 than in the WT (Figure 1B). The distance
between Tyr156 and Leu195 also indicated that the proportion
of open state in seq1/seq5 was much more occupied than in the
WT during the simulations (Figure S3). The prolonged open
state of the pocket induced by mutations may be the main
Although a more open entrance conformation was achieved by seq1
than seq5, the activity of the single point mutant seq1 remained low.
The expanded pocket caused by Y190P (seq1) provided basic and
essential space for CPPK access, which resulted in detectable activity
compared with WT. However, seq1 bound CPPK with relatively poor
affinity compared to seq5 (binding free energy calculated as -25.3 ±
2.9 vs. -18.1 ± 2.3 kcal·mol-1). This suggests that the other four
mutations contribute significantly to the enhanced affinity and
activity in seq5. As illustrated in Figure 2A, the mutations in seq5
contributed to binding through different interactions.
I144V/E145C located in the catalytic loop and
Y190P/L199V/M206F located in the binding loop form the
expanded pocket. They interact with substrates through
energy-favouring interactions. In this conformation, two
hydrogen bonds between O1CPPK and catalytic Try156/Ser143
are formed. Y190P interacts with CPPK via a π-alkyl interaction.
A π-sulphur interaction was observed between E145C and the
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two phenyl groups. Moreover, the phenyl groups of CPPK chlorine-substituted ketones. This indicates the importance of
View Article Online
DOI: 10.1039/C9CY02444A
interacted with M206F and Tyr249 in π-π stacking with a T chlorine position, which may affect substrate binding by
shape, as was expected. The π-alkyl interaction observed noncovalent interaction. The expanded substrate scope further
between the Leu195 and L199V side chains and CPPK may confirms that the engineered binding pocket reduced the
synergistically influence CPPK binding. In addition, the amide hindrance near catalytic residues, which demonstrated a
oxygen of Leu195 forms a halogen bond with the para-chlorine general increase in enzyme activity in different substrates.
of CPPK. These interactions favour substrate binding and anchor
the phenyl in the expanded pocket, which dramatically Investigation of stereocontrol elements in mutant seq5
increases the activity of LkADH. Therefore, in addition to the
more open entrance, the favourable interactions between the
substrate and residues that help stabilise substrate binding are
also crucial for activity enhancement.
Figure 4. Binding orientation determined the stereoselectivity. ADPR, adenosine
diphosphoribose. Based on the Prelog rule for predicting the stereo-preference of
alcohol dehydrogenases (ADHs), the hydride from the coenzyme (NADH/NADPH)
can attack the carbonyl of an asymmetrical ketone from either the Re- or Si-face to
produce (R)- or (S)-products, respectively. In our study, the CPPK was expected to
bind as pro-R pose.
Steric hindrance, the determining factor of stereoselectivity in
WT, was reduced in seq5. Due to the excellent stereoselectivity
toward CPPK, it could infer that a different stereocontrol
element played a role in seq5. Based on the Prelog rule of chiral
recognition of ADHs3, 25-28, the stereoselectivity of LkADH was
determined by the binding orientation of the substrate (Figure
4). The subtle structural differences between the two aromatic
rings of CPPK renders them extremely challenging targets for
asymmetric catalysis. Therefore, for the substrate CPPK, in
Figure 3. Activity determination of bulky-bulky and bulky-small ketones. (A) Structures
order to transfer hydride from the Si-face, the binding pocket
should be able to discriminate para-chlorine-substituted phenyl
and non-substituted phenyl to prefer pro-R binding, which was
key for the high stereoselectivity to CPPK. It is reasonable to
presume that para-chlorine affects substrate binding
orientation, and thus, stereoselectivity. Therefore, to explore
the impact of chlorine substitution, the stereoselectivities of
seq5 toward chlorine-substituted ketones with different
positions (1a-3a; 7a-9a) and non-substituted ketones (4a, 6a)
were determined (Figure 5).
All bulky-small ketones were reduced in R enantiomer by WT,
and three of them obtained with a relatively high ee of >99%.
The 2-chloro-1-phenylethanone (10a) obtained a relatively low
ee of 65%. These results indicate that WT was an anti-Prelog-
selective ADH that produced (R)-enantiomers, which is
consistent with a previous report29. The small and large binding
pockets differ in size in the WT, which fixes the substrate-
binding orientation. As a result, the substitutions on the bulky
of small-bulky and bulky-bulky ketones used in this study. (B) Comparison of specific
activities of seq5 and WT.
We further compared the specific activities of the WT and seq5
to nine ketones. The specific activities for bulky-bulky ketones
(1a-5a) and bulky-small ketones (6a-9a) are compared in Figure
3. Compared to the WT, engineered seq5 demonstrated higher
activity in all tested substrates. Substrate 5a demonstrated the
highest activity of seq5 among all tested ketones. Its
corresponding chiral alcohol, (S)-(4-chlorophenyl)(pyridin-2-
yl)methanol (CPMO), is an important intermediate in the
synthesis of the antihistamine drugs bepotastine and
carbinoxamine24. Meanwhile, the WT has no measurable
activity toward 1a-5a (specific activity less than 0.001
µmol·min−1·mg−1) and was only active toward small-bulky
ketones. Furthermore, compared to ketones without
substitution, electron-withdrawing chlorine substitution
seemed to enhance activity. Moreover, seq5 demonstrated
gradually increasing activities from ortho-chlorine- to para-
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group have almost no impact on stereoselectivity, as steric was absent from the pro-S docking pose (Figure 2; FViiegwuArreticSle5Oanlninde
DOI: 10.1039/C9CY02444A
hindrance mainly determines the stereoselectivity.
S6).
Figure 6. Opposite binding orientation of 2-chloro-1-phenylethanone and 4-chloro-1-
phenylethanone in seq5. It was controlled by the different halogen bonds which led to
stereoselectivity inversion. 2-chloro-1-phenylethanone and 4-chloro-1-
phenylethanone are represented in cyan and orange, respectively. Halogen bonds are
represented in blue; π-sulfur interactions in orange; π-π interactions in purple;
hydrogen bonds in green. A. 4-chloro-1-phenylethanone bound in pro-R pose.
Halogen bonds formed between para-chlorine and Leu195. B. 2-chloro-1-
phenylethanone bound in pro-S pose. Halogen bond formed between ortho-chlorine
and Gly189 or Pro188. C. Overlay of the two substrate conformations, which bound in
opposite orientation.
Figure 5. Stereoselectivities of seq5 to different substituted ketones. The chlorine
position plays an important role in product stereo configuration.
Surprisingly, contrary to the strict anti-Prelog-selectivity of the
WT, the stereoselectivity of seq5 was inverted toward some
substrates, demonstrating an opposite stereo-preference. Seq5
was highly anti-Prelog selective to para-chlorine-substituted
ketones, such as 3a (CPPK), 5a, and 9a, all producing (R)-
enantiomers, except 5a. The 5a was reduced to (S)-enantiomer
due to the presence of a pyridine group instead of phenyl, which
is higher than a large phenyl group according to the Cahn–
Ingold–Prelog (CIP) rule. However, the stereoselectivities
inverted to Prelog selective when 2-chlorodiphenylketone (1a),
Very recently, during the engineering of KpADH from
Kluyveromyces polyspora, high stereoselectivity was obtained
using substrate 5a due to the opposite charge characteristics
between chlorophenyl and pyridine substitutes24. KpADH was
revealed to prefer the formation of the pro-R pose of 5a
because of the electrostatic attraction formed between the
positively charged pyridine substitute and the negatively
charged Glu21424. In the present study, no charged residues
interacted with any substrates, which suggests a different chiral
recognition mechanism in seq5. The halogen bond is commonly
found in protein-ligand complexes, with >2000 structures
having been reported in recent years30. It is analogous to
hydrogen bonds and is highly directional and specific31. It is also
applied to control substrate selectivity in biocatalysis32. In
addition, although halogen bonds have been successfully
2-chloro-1-phenylethanone
(7a),
and
3-chloro-1-
phenylethanone (8a) were reduced, mainly yielding (S)-
enantiomers. Therefore, docking experiments were performed
using 4-chloro-1-phenylethanone (9a) producing (R)-
enantiomer of 82% ee and 2-chloro-1-phenylethanone (7a)
producing (S)-enantiomer of >99% ee, which may reveal the
reason for stereoselectivity inversion (Figure 6). The 4-chloro-1-
phenylethanone bound in pro-R pose, the para-chlorine of
which formed a halogen bond with Leu195, stabilising the pro-
applied in enantioseparations and chemical chiral catalysts33-35
,
R
pose (Figure 6A). On the contrary, the 2-chloro-1-
to the best of our knowledge, the role of the halogen bond in
determining stereoselectivity has not been reported in the area
of biocatalysis. All the results we obtained indicate that the
elimination of steric hindrance in seq5 created sufficient space
and appropriate conformation for phenyl ring inversion and
new noncovalent bonds formation. In this situation, differences
in the sizes of groups had a limited impact on stereoselectivity,
and the noncovalent halogen bond mainly determined the
stereoselectivity. The main stereocontrol element changed in
LkADH after engineering.
Based on the results above, seq5 prefers the pro-R binding of
CPPK mainly because of the halogen bond between the para-
chlorine and oxygen of Leu195. Moreover, the absence of a
halogen bond with a pro-S conformation may cause difficulty in
catalysis initiation. The catalysis mechanism of ADH revealed
that hydride transfer can happen when the distance between
the NADPH-C4 atom and ketone carbonyl carbon is ≤ 4.5 Å, and
the carbonyl oxygen forms a hydrogen bond with the side
chains of the tyrosine catalytic residue24, 36-41. Therefore, the
CPPK conformations in which the carbonyl oxygen formed
hydrogen bond interactions (≤ 3.7 Å) with Tyr156 and the
distance between the carbonyl carbon and NADPH-C4 atoms
was less than 4.5 Å were defined as pre-reaction conformations.
phenylethanone bound in pro-S pose (Figure 6B), the phenyl
ring of which was located in the expanded pocket and
interacted with Y249 and F206 as a π-π T-shape. Significantly,
the ortho-chlorine had halogen bonds with Gly189 and Pro188.
The superimposition clearly illustrated the opposite binding
orientation of two substrates, resulting in opposite enantiomers
(Figure 6C). Similar binding conformations of 2-
chlorodiphenylketone (1a) producing (S)-enantiomer of 57% ee
and CPPK were also observed (Figure S4). These results further
revealed that sufficient space was created for the inversion of
binding orientation and the halogen bond controls the binding
orientation in seq5.
In addition, it should be noted that ketones without any
substitution on phenyl, such as phenyl-2-pyridinylmethanone
(4a) and acetophenone (6a), only reduced with a low ee of 7%
and 35.9%, respectively. Whereas when para-chlorine was
attached, the ee of 5a and 9a dramatically increased to >99%
and 82%, respectively. These significant increases and the
inversion in stereoselectivity clearly indicate that the halogen
bonds formed by chlorine substitution play an important role in
the determination of stereoselectivity. This important halogen
bond was also observed in the pro-R docking pose of CPPK but
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Both the docking conformations of the pro-R and pro-S pose reaction conformations more easily than the VipewroA-rtSiclepOonslinee,
DOI: 10.1039/C9CY02444A
were pre-reaction conformations (Figure 7A, 7C). However, resulting in (R)-CPPO with high ee.
seq5 only produced an (R)-enantiomer, and previous reports
have shown that docking analysis alone may create an Scale-up preparation of (R)-CPPO and (S)-CPMO
incomplete picture of ligand binding42.
With the highly stereoselective seq5, we scaled-up the
production (200 mL) of (R)-CPPO and (S)-CPMO to determine
the practical application of the engineered biocatalyst. Results
of the scaled-up production demonstrate that both valuable
diaryl alcohols can be easily prepared with high ee and complete
conversion. The (R)-CPPO concentration was calculated as 87.5
g/L, and the (S)-CPMO was calculated as 87.8 g/L. The 200 mL
reaction produced 15.3 g of (R)-CPPO with an ee of 99.3% and
an isolated yield of 88.1%. The CPPO was confirmed by NMR
analysis: 1H-NMR(600MHz，CDCl3) δ：7.33(4H, d, J = 4.28 Hz),
7.27-7.29(5H，m), 5.78 (1H，s)；13C-NMR(150MHz，CDCl3) δ
：143.4, 142.2, 133.3, 128.7, 128.7, 128.6, 128.6, 127.9, 127.9,
127.8 127.8, 126.5, 75.6. The chiral HPLC chromatograms of (R)-
CPPO after a reaction period of 16 h are shown in Figure S7. The
200 mL reaction produced 14.8 g of (S)-CPMO with an ee of
98.1% and an isolated yield of 84.2%. The CPMO was confirmed
by NMR analysis: 1H-NMR(600MHz，CDCl3) δ：8.55(1H, d, J =
4.92 Hz), 7.62(1H, ddd, J = 15.36, 7.62, 1.68 Hz), 7.29-7.32(4H，
m), 7.20(1H, dd, J = 7.02, 5.16 Hz), 7.12(1H, d, J = 7.86 Hz), 5.73
(1H ， s) ； 13C-NMR （ 150MHz ， CDCl3 ） δ ： 160.4, 147.9,
Figure 7. Comparison of two critical distances between the pro-R and pro-S pose. The
yellow dashed line represents the initial distance obtained in docking results. The
deeper red color represents the higher probability of corresponding conformations. A.
Docking result obtained for the pro-R pose. B. The probability density distribution of
141.7, 137.0, 133.6, 128.7, 128.7, 128.4, 128.4, 122.6, 121.3,
74.3.
Among the variants recently reported, seq5 demonstrated an
attractive substrate tolerance and satisfactory ee without
additional optimisation (Table S2). These results demonstrate
that the bio-reduction approach using seq5 has potential for
further large-scale applications.
pro-R conformations obtained from the simulations. The conformations of pro-R
binding demonstrated a close contact to NADPH and Tyr156 simultaneously. C.
Docking result obtained for the pro-S pose. D. The probability density distribution of
pro-S conformations obtained from the simulations. The conformations of pro-S
binding demonstrate relatively dispersed distances to Tyr156 and NADPH.
Considering the dynamic motion of the substrate-enzyme
complex, MD simulation was performed to further analyse the
two critical distances and compare the proportions of pre-
reaction conformations of the pro-R and pro-S poses. As
previous reports suggesting multiple short simulations achieve
better correlation of stereoselectivity than a single long
simulation43, 44, the two distances of the pro-R and pro-S
conformations were monitored with ten independent 4 ns
simulations (in total 40 ns). The binding free energy of the pro-
S pose was calculated as -22.1 ± 2.5 kcal·mol-1, higher than the
pro-R pose of -25.3 ± 2.9 kcal·mol-1. Furthermore, as illustrated
in Figure 7B, the pro-R conformations of CPPK demonstrated a
close and relatively stable distance with NADPH and Tyr156.
However, it is difficult for the pro-S pose to sustain stable close
contact with NADPH and Tyr156, which implies that the
probability of hydride transfer from the Re-face was quite low
(Figure 7D). Remarkably, the proportion of pre-reaction
conformations with both distance C4NADPH-C1CPPK ≤ 4.5 Å and
distance OHTyr156-O1CPPK ≤ 3.7 Å was 22.2% in the pro-R pose and
7.3% in the pro-S pose. The occurrence of more than three
times of pre-reaction conformations of the pro-R pose than
those of the pro-S pose indicated that seq5 prefers to transfer
hydride from the Si-face producing (R)-CPPO, which is
consistent with the bio-reduction results. The above results
suggest that the pro-R pose with a halogen bond can form pre-
Conclusions
In this study, we created an ADH variant with enhanced activity
and stereoselectivity toward diaryl ketone without property
trade-offs and investigated the molecular basis. A “shrinking
mutagenesis” strategy was proposed to screen mutants with
increased activity and stereoselectivity. The obtained seq5
could reduce the bulky-bulky model substrate CPPK and
generate an (R)-enantiomer with a high ee of >99%. The activity
and stereoselectivity were further investigated using five bulky-
bulky ketones and four bulky-small ketones. Seq5 had higher
activity than the WT toward all nine tested substrates.
Molecular docking and MD simulations revealed that the
original binding pocket was dramatically expanded, not only
because of a smaller side chain but also the more open binding
loop induced by Y190P. Therefore, the change in the
preorganisation state of seq5 facilitated the approach of CPPK
to catalytic residues. Moreover, all five constructed mutations
provided energy favourable interactions and facilitated
substrate binding in the pro-R pose. In the WT, stereoselectivity
was mainly determined by steric effects. However, in seq5, after
the elimination of steric hindrance, the main stereocontrol
element probably changed to the halogen bond. The MD
simulations further revealed that the formation of pre-reaction
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19 X. D. Kong, S. Yuan, L. Li, S. Chen, J. H. Xu and J. Zhou, PNAS
conformation would be easier with the pro-R pose with a
halogen bond and π-π interaction than with the pro-S pose.
These results indicate that the stereocontrol element of LkADH
was changed to recognise diaryl ketones. To avoid property
trade-offs, changes of stereocontrol elements could be
important for the simultaneous enhancement of activity and
stereoselectivity in ADH. Furthermore, the obtained seq5 is a
promising biocatalyst for the preparation of valuable chiral
diaryl alcohols.
View Article Online
USA, 2014, 111, 15717.
DOI: 10.1039/C9CY02444A
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This work was supported in part by grants from the National
Natural Science Foundation of China (No. 81773616), Program
of Shanghai Technology Research Leader (No. 17XD1423200)
and the Seed Fund Program of the Shanghai University of
Medicine & health Sciences (SFP-18-22-07-002). We are also
grateful to professors Jiahai Zhou and Hualei Wang for fruitful
discussions.
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Catalysis Science & Technology
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DOI: 10.1039/C9CY02444A
Table of contents entry
Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity
toward diaryl ketones: reduction of steric hindrance and change of stereocontrol element
Graphical abstract