Conformational Dynamics-Guided Loop Engineering of an Alcohol Dehydrogenase: Capture, Turnover and Enantioselective Transformation of Difficult-to-Reduce Ketones

Article abstract of DOI:10.1002/adsc.201900249

Directed evolution of enzymes for the asymmetric reduction of prochiral ketones to produce enantio-pure secondary alcohols is particularly attractive in organic synthesis. Loops located at the active pocket of enzymes often participate in conformational changes required to fine-tune residues for substrate binding and catalysis. It is therefore of great interest to control the substrate specificity and stereochemistry of enzymatic reactions by manipulating the conformational dynamics. Herein, a secondary alcohol dehydrogenase was chosen to enantioselectively catalyze the transformation of difficult-to-reduce bulky ketones, which are not accepted by the wildtype enzyme. Guided by previous work and particularly by structural analysis and molecular dynamics (MD) simulations, two key residues alanine 85 (A85) and isoleucine 86 (I86) situated at the binding pocket were thought to increase the fluctuation of a loop region, thereby yielding a larger volume of the binding pocket to accommodate bulky substrates. Subsequently, site-directed saturation mutagenesis was performed at the two sites. The best mutant, where residue alanine 85 was mutated to glycine and isoleucine 86 to leucine (A85G/I86L), can efficiently reduce bulky ketones to the corresponding pharmaceutically interesting alcohols with high enantioselectivities (～99% ee). Taken together, this study demonstrates that introducing appropriate mutations at key residues can induce a higher flexibility of the active site loop, resulting in the improvement of substrate specificity and enantioselectivity. (Figure presented.).

Full text of DOI:10.1002/adsc.201900249

Title: Conformational Dynamics-Guided Loop Engineering of an
Alcohol Dehydrogenase: Capture, Turnover and Enantioselective
Transformation of Difficult-to-Reduce Ketones
Authors: Beibei Liu, Ge Qu, Junkuan Li, Wenchao Fan, Jun-An Ma,
Yan Xu, Yao Nie, and Zhoutong Sun
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900249
Link to VoR: http://dx.doi.org/10.1002/adsc.201900249

Advanced Synthesis & Catalysis
10.1002/adsc.201900249
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Conformational Dynamics-Guided Loop Engineering of an
Alcohol Dehydrogenase: Capture, Turnover and
Enantioselective Transformation of Difficult-to-Reduce Ketones
+
a,b
+b
b,c
b
c
a
Beibei Liu, Ge Qu, Jun-Kuan Li, Wenchao Fan, Jun-An Ma, Yan Xu, Yao
a,*
b,*
Nie, and Zhoutong Sun
a
School of Biotechnology, Key laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University,
Wuxi 214122, China
(
+86) 510-851987760; e-mail: ynie@jiangnan.edu.cn
b
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport
Economic Area, Tianjin 300308, China
(
+86) 22-84861981; e-mail: sunzht@tib.cas.cn
c
Department of Chemistry, Tianjin Key Laboratory of Molecular Optoelectronic Sciences, and Tianjin Collaborative
Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China
These two authors contributed equally to this work.
+
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. Directed evolution of enzymes for the asymmetric region, thereby yielding a larger volume of the binding pocket
reduction of prochiral ketones to produce enantio-pure to accommodate bulky substrates. Subsequently, site-directed
secondary alcohols is particularly attractive in organic synthesis. saturation mutagenesis was performed at the two sites. The
Loops located at the active pocket of enzymes often participate best mutant, where residue alanine 85 was mutated to glycine
in conformational changes required to fine-tune residues for and isoleucine 86 to leucine (A85G/I86L), can efficiently
substrate binding and catalysis. It is therefore of great interest to reduce bulky ketones to the corresponding pharmaceutically
control the substrate specificity and stereochemistry of interesting alcohols with high enantioselectivities (~99% ee).
enzymatic reactions by manipulating the conformational Taken together, this study demonstrates that introducing
dynamics. Herein, a secondary alcohol dehydrogenase was appropriate mutations at key residues can induce a higher
chosen to enantioselectively catalyze the transformation of flexibility of the active site loop, resulting in the
difficult-to-reduce bulky ketones, which are not accepted by the improvement of substrate specificity and enantioselectivity.
wildtype enzyme. Guided by previous work and particularly by
structural analysis and molecular dynamics (MD) simulations, Keywords: directed evolution; conformational dynamics;
two key residues alanine 85 (A85) and isoleucine 86 (I86) alcohol dehydrogenase; activity; enantioselectivity
situated at the binding pocket were thought to increase the
fluctuation of a loop
and (S)-configurated alcohols. Whenever stereoselectivity
does not meet all requirements for an ecologically and
Introduction
economically viable process, directed evolution can be
Enantio-pure alcohols are essential building blocks for
[
2]
invoked. ADH-based biocatalysis is thus complementary
to chiral transition metal catalyzed reduction of prochiral
the preparation of chiral therapeutic drugs and other fine
chemicals which is the reason that catalytic asymmetric
reduction of prochiral ketones to optically pure alcohols
continues to be an important issue in synthetic organic
chemistry. The two options are transition metal catalysis or
[
3]
ketones.
As
an
attractive
ADH
in
biotechnology,
Thermoanaerobacter brockii alcohol dehydrogenase
TbSADH) is a promising biocatalyst employed in a series
(
[
1]
biocatalysis using enzymes. The former has been shown
to be successful in numerous academic and industrial
studies, but challenges remain when so-called difficult-to-
reduce ketones are involved, specifically those in which
the α- and α'-substituents flanking the carbonyl moiety are
sterically similar, or those that are characterized by two
bulky aryl groups (diaryl ketones). Alternatively, as natural
biocatalysts, alcohol dehydrogenases (ADHs, EC 1.1.1.X,
X = 1 or 2) are capable of reducing prochiral ketones under
mild conditions. The reduction of prochiral ketones by
ADHs is often reported as green routes to access both (R)-
of synthetic applications due to its high thermostability and
pronounced stereoselectivity in the reduction of many
[
3a,4]
structurally different ketones.
medium-chain dehydrogenase/reductase (MDR) family.
TbSADH belongs to the
[
5]
In ketones reduction, the carbonyl oxygen of the substrate
coordinates to the Zn2+ and a hydride is supplied from the
[
6]
cofactor NADPH to the carbonyl C atom of the substrate.
The crystal structure of TbSADH (Figure S1a) has been
solved at 2.5 Å resolution and shows that the substrate
binding sites are composed of two hydrophobic pockets,
[
7]
one smaller than the other (Figure S1b). The distinct
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201900249
sizes of the two pockets control the stereochemical identity
of the product (Figure S1c), obeying Prelog’s rule in
which NADPH delivers its pro-R hydride to the Re face of
a ketone, producing (S)-configured alcohols (Figure S1d).
In fact, previous studies have demonstrated that
performing site-specific mutagenesis on either single or
combined residues lining the binding pocket can efficiently
affect the enantioselectivity, substrate specificity and other
and M285 lining the binding pocket were also included
[
6a]
(Figure S2). Residues A85, I86 and W110 are located in
the same loop region (loop A), and Y267 occurs in loop B,
whereas L294 and C295 are located in another loop (loop
C) (Figure S2). Other residues, including C37, H59 as
well as D150, coordinately bonded to zinc atom, are not
considered in this study. In order to monitor the structure
flexibility of the above mentioned residues lining the
binding pocket, 100 ns MD simulations on the WT enzyme
were performed at 30 and 60 °C, respectively. The analysis
of the Root Mean Square Fluctuations (RMSFs) allowed us
to identify the flexible regions representing more than 1 Å
fluctuation. As shown in Figure 1 and Figure S3, the four
residues A85, I86, L294, and C295 were relatively rigid,
indicating that the less flexible residues may impose
restrictions on substrate recognition.
[
8]
properties of TbSADH.
In recent years, there has been growing interest in the
conformational dynamics of proteins, which play an
important role in enzyme catalysis, and engineering of
enzymes guided by conformational dynamics has become
an effective strategy for protein evolution, achieving
[
9]
successes in expanding substrate scope,
increasing
[
10]
[11]
enantioselectivity,
relieving product inhibition,
and
[
12]
improving thermostability.
The motion of amino acid
residues in loops involved in the pocket has been
recognized to influence the catalytic properties of
enzymes.[
9,13]
Although the residues in loops generally do
not act as catalytic sites, they often participate in
conformational changes required for fine-tuning residues
[
14]
that are relevant to substrate binding and catalysis.
Moreover, conformational dynamics influencing the
substrate scope of ADHs were explored in previous
studies,[
6b,8d]
which inspires us to extend this subject to the
Figure 1. Average RMSF values of all residues calculated
from MD simulations for the apo WT enzyme at 30 °C and
difficult-to-perform asymmetric reduction of bulky ketones
such as 1 with formation of (S)-2 (Scheme 1). (S)-2 is a
crucial chiral synthon needed in the preparation of
6
0 °C.
[
15]
antiallergic
drugs
Bepotastine
and
(S)-
[
16]
Carbinoxamine. The challenging asymmetric reduction
of prochiral diaryl ketones in general has attracted much
attention in pharmaceutical application. Unfortunately,
wildtype (WT) TbSADH failed to catalyze this kind of
reaction, and the few biocatalysts (Table S1) that were
On the basis of the crystal structure 1YKF, all of the
four relatively rigid residues are located at the first shell of
the substrate binding pocket, and the distances between the
zinc ion to the Cα atoms of each residue are 7.2 Å (A85),
9
.7 Å (I86), 10.5 Å (L294) and 9.5 Å (C295), respectively.
reported to reduce ketone 1 to (S)-2 did so with insufficient
To understand how possible mutations of the key residues
manipulate loop fluctuation, I86, L294, and C295 were in
silico substituted with alanine in terms of alanine scanning,
whereas A85 was replaced by glycine. Then 100 ns MD
simulations were carried out for the four variants,
respectively. As a result, the RMSFs of A85G and I86A
showed clear changes within loop A, compared to the WT
activity.[
17]
Herein, we show that the conformation
dynamics-guided loop engineering strategy leads to a
TbSADH mutant with excellent enantioselectivity (>99%
ee) and catalytic efficiency (703.52 s- mM ). We also
demonstrate that other sterically demanding substrates can
likewise be reduced with high activity and
enantioselectivity. In terms of understanding the results on
a molecular level, molecular dynamics (MD) simulations
throw light on the origin of enhanced activity and
selectivity.
1
-1
(
Figure S4). In contrast, L294A and C295A did not show
any distinct fluctuations within loop C (Figure S4),
indicating that substitutions on A85 and I86 are more
likely to trigger conformational dynamics.
Site-directed saturation mutagenesis of TbSADH
inert residues in loops. Based on the above structural
analysis and MD simulations, two key inert residues A85
and I86 emerged as possible sites for mutagenesis leading
to more fluctuating loops. Therefore, the two residues were
subjected to saturation mutagenesis (SM), which is a
powerful mutagenesis strategy for protein engineering and
Scheme 1. ADH-catalyzed asymmetric reduction of ketone
1
.
[
18]
directed evolution. The two sites were first individually
substituted to the other 19 canonical amino acids by the
Results and Discussion
[
19]
Tang method.
Accordingly, a set of four degenerate
Conformational dynamics-guided identification of key
residues at the binding pocket. Our previous study
revealed that residues A85, I86, W110, L294 and C295 are
codons of NDT, VMA, ATG, and TGG were designed for
encoding all 20 amino acids without introducing
redundancy, and requiring in each case the screening of 60
[
4c]
involved in tuning enantioselectivity and activity.
We
[
20]
transformants for 95% library coverage. By screening
the two independent libraries, a few mutants with activity
towards ketone 1 were indeed obtained, including A85G,
therefore selected them as potential candidates for
mutagenesis.[ In addition, based on the crystal structure of
8]
[
7]
TbSADH (PDB code 1YKF), the residues S39, Y267
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Advanced Synthesis & Catalysis
10.1002/adsc.201900249
I86A, I86S, I86E, I86T, I86L, and I86P, all favoring (S)-2,
except for I86P (Table 1).
WT
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
-
-
-
A85G/I86A
A85G/I86V
A85G/I86L
A85G/I86C
A85L/I86C
A85V/I86C
A85S/I86C
A85S/I86L
A85L/I86L
A85C/I86S
A85S/I86S
A85V/I86S
76
24
98
61
12
3
5
7
3
67
9
>99(S)
93(S)
>99(S)
98(S)
88(S)
Table 1. Results of saturation mutagenesis of single
residues using NDT/VMA/ATG/TGG as degenerate
codons.
c(%)a
-
6
13
2
13
6
ee(%)b
-
c
Code
WT
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
Mutation
-
-
82(S)
89(S)
A85G
I86A
I86Q
I86S
I86E
I86G
I86T
I86L
I86P
I86C
I86R
I86K
96(S)
80(S)
c
-
c
>99(S)
84(S)
>99(S)
-
93(S)
81(S)
90
c
a)
4
-
Conversion (c%) was determined by HPLC analysis as
b)
16
10
6
1
2
94(S)
90(S)
10(R)
shown in the Supporting Information. The ee values were
determined by chiral HPLC analysis; the absolute
configuration was determined by comparison of the elution
order with literature data (see the Supporting Information).
c
-
c
c)
-
ee value was not determined if conversion is lower than
c
1
-
5%.
a)
Conversion (c%) was determined by HPLC analysis as
shown in the Supporting Information. b) The ee values
were determined by chiral HPLC analysis; the absolute
configuration was determined by comparison of the elution
order with literature data (see the Supporting Information).
Kinetic
parameters,
thermostability,
and
preparative-scaled reactions. The best TbSADH mutants
were then characterized by enzyme kinetics and
thermostability assays using purified proteins. As shown in
c)
ee value was not determined if conversion is lower than
Table 3, T15 and T24 variants showed similar K
and consequently led to comparable catalytic efficiency
) toward cofactor NADPH. However, for ketone 1,
m
and kcat,
5
%.
(kcat/K
m
-
1
T15 showed a higher turnover number (kcat 37.99 s ) and
lower substrate affinity (K 0.054 mM) compared to T24,
and represented the best catalytic efficiency, where the
In order to further optimize enantioselectivity and
improve activity in the reaction of ketone 1, a highly
focused library combining A85 and I86 with reduced
alphabets was generated using the Combinatorial Active-
m
-
1
-1
k
cat/K
m
value even reaches 703.52 s mM . Concerning the
site Saturation Test (CAST) concept.[
2b,21]
issue of enzyme robustness of the two variants,
Based on Table
15
thermostability was assessed by measuring T50 values, the
temperature at which 50% of enzyme activity is lost
following a heat treatment for 15 min. Both variants show
1
, only the amino acid alphabets with small steric
hindrance (including A, I, L, G, S, C, T and V) were used
as building blocks to perform SM at the two positions
simultaneously, and the appropriate degenerate codons of
DYA and KGC were designed to reduce screening effort
so that only 192 transformants had to be screened for 95%
library coverage. The results showed that both conversions
and ee-values were highly improved by the double mutants
1
5
T
50 values that exceed 67 °C. The results of kinetic
studies are summarized in Table 3. Subsequently, the
preparative scale reactions for the selected variants were
performed using 50 mM of ketone 1 (1.09 g) in 100 mL of
reaction volume, with 10% isopropanol as cosolvent and as
assistance for cofactor regeneration (Figure S6). As the
T15 variant shows the best performance, it was then
evaluated in a higher concentration of ketone 1 (100 mM,
(
Table 2). T15 (A85G/I86L) showed the best performance,
successfully transforming ketone 1 to (S)-2 with 99% ee at
8% conversion (Figure S5).
9
2
.17 g) in 100 mL of reaction volume. Full conversion was
Table 2. Results of combinatorial mutagenesis of residues
A85 and I86 using DYA/KGC as degenerate codon.
achieved within 4 h (Figure S7), which maintaining the
originally evolved high enantioselectivity of 99% ee with
9
3% isolated yield.
Code
Mutation
c(%)a
ee(%)b
Table 3. Kinetic parameters and thermostability of TbSADH mutants for ketone 1.
1
NADPH
Thermostability
Mutant
K
mM)
0.05±0.01
0.10±0.01
m
k
(s )
cat
k
cat/K
m
-1
K
(mM)
0.075±0.01
0.076±0.01
m
k
(s )
cat
-1
kcat/K
m
(s mM )
553.91
1
5
T50 (°C)
-
1
-1
-1
-1
(
(s mM )
703.52
219.71
T15
T24
67
68
37.99±0.43
22.85±0.46
41.82±1.68
35.37±1.23
465.39
Shedding light on the origin of conformational
dynamics. In order to provide insights into how the
mutations at the two sites endow the best mutant T15 with
dramatically enhanced activity towards ketone 1, we
analyzed in more detail the structures from the MD
simulations performed of the apo state for the T15 variant
in comparison with the WT enzyme. The T15 variant was
found to have a conformation similar to that of the WT
enzyme (PDB code 1YKF, chain A), except for the
positions 85 and 86 (Figure 2a). A main-chain motion was
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Advanced Synthesis & Catalysis
10.1002/adsc.201900249
observed at the both sites, 1.9 Å and 0.9 Å for the positions
8
5 and 86, respectively (Figures 2b and 2c).
Figure 2. Overlay of representative snapshots for the
catalytic pocket. (a) The small pocket of T15 (in cyan
color) and the WT enzyme (PDB code 1YKF, in grey
color). Steric hindrance of the side-chains of A85 (b) and
I86 (c) is indicated by red dashed cycle. The red arrows
and distances (in angstroms) indicate the motion of the Cα
atoms of residues 85 and 86.
Figure 3. Overlay of representative structures for T15
variant (a) and WT (b). The inset depicts the deviation of
residues 85 and 86 during the 100 ns MD simulations.
Moreover, to investigate and compare the
conformational changes of the T15 variant relative to WT,
cluster analysis was conducted on the 100 ns MD
trajectories, using the hierarchical agglomerative (bottom-
Following the reduction mechanism of TbSADH, the
carbonyl oxygen of the substrate coordinates to the Zn
and a hydride is supplied from NADPH to the carbonyl
carbon atom, and the binding orientation of the substrate
2+
[
22]
up) approach
for grouping similar molecular
configurations into distinct sets. An overlay of ten
representative structures from cluster analysis in terms of
RMSD cutoff is represented in Figure 3. In sharp contrast,
a higher loop movement and less steric hindrance at
residues 85 and 86 compared to WT was observed in the
T15 variant (Figure 3 and S8), indicating that introducing
mutations at residues 85 and 86 can improve the flexibility
of the active site loop. Indeed, it has been demonstrated
that glycine in loops can influence loop dynamics
locally.[ For variant T15, the volume of the binding
with respect to the NADPH determines the stereochemistry
[4c, 6b]
of the product.
In order to explore the effect of
mutation on stereoselectivity at the molecular level,
docking and MD calculations were performed. For the T15
variant, the preferred binding mode of ketone 1 results in
non-bonded interactions between the two bulky substituent
groups and the side chains of residues, including π-π
interactions between pyridyl of 1 and W110, as well as
phenyl of 1 and Y267, hydrogen bonding and hydrophobic
interactions (Figure S10a). For these existing interactions,
the substrate preferentially orientates such that the π-face
of the C=O bond undergoes nucleophilic attack to yield
23]
3
3
pocket is expanded to 213±41 Å , compared to 141±33 Å
of WT. Such noticeable differences are responsible for
enhancing loop fluctuation and decreasing the steric
hindrance induced by the double mutations (A85G/I86L).
This confers the binding pocket higher plasticity and thus
the ability to accept bulky non-natural substrates, such as
ketone 1. Although both of the large and small binding
pockets are expanded after mutation (Figure 2), it’s
interesting to note that the volume of the small pocket (at
the 85G/86L side) is increased much more visible.
(
S)-2. During the MD simulations of the T15 complex, the
hydride transfer distance and the pro-(S) angle between the
nucleophile and the electrophile range mainly from 120° to
1
60°, and 3.5 Å to 4.5 Å, respectively (Figure S10b),
indicating that the docked pro-(S) pose of 1 shown in
Figure S10a is favored in accord with variant T15, which is
consistent with the experimental stereochemical result.
Testing the best mutants as catalysts in the reduction
of other bulky ketones. The T15 and T24 variants
evolved for ketone 1 were then tested as catalysts in the
asymmetric reduction of a series of diaryl ketones to
explore the substrate scope. Compounds 3a~3f consist of a
pyridine ring and a benzene substituent, while 3g and 3h
These crucial effects are not observed at the other
residues lining the binding pocket, as shown by the
comparable geometric parameters of T15 variant and WT
(
8
Figure S9). Clearly, only the double mutations at hotspots
5 and 86 influence flexibility at the binding pocket.
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Advanced Synthesis & Catalysis
10.1002/adsc.201900249
are composed of two benzene groups (Figure 4). Variants
T15 and T24 can catalyze most of them with excellent
enantioselectivity and conversions, except for 3d which is
not accepted by T24. Moreover, WT shows no activity
toward 3b and 3d, which can be transformed by T15,
suggesting that T15 has a wider substrate scope. It is
interesting to note that 3a and 3f are reduced to the (S)-
alcohol by WT, but the variants induce opposite
stereoselectivity. We speculate that this might be caused by
intermolecular forces (e.g., van der Waals force) nearby
the loop region containing residues 85 and 86, where
negative electrostatic potential was detected (Figure S11).
In order to gain insight whether such a drastic change of
stereoselectivity and activity is general, we also performed
the reduction of aliphatic (3i~3k) and aryl ketones (3l~3n)
discovered that indeed reduce ketone 1 with unusually high
(S)-selectivity amounting to 99% ee, without significant
tradeoff in thermostability. In particular, the T15 variant
displayed the highest catalytic efficiency toward ketone 1
-
1
-1
with kcat/K
m
of 703.52 s mM . Importantly, molecular
dynamics (MD) simulations unveiled the origin of loop
conformational dynamics, and demonstrated that
introducing appropriate mutations at residues 85 and 86
induces a higher flexibility of the active site loop, thereby
conferring the ability to change the shape of the binding
pocket and consequently to catalyze the reaction of
difficult-to-reduce ketones.
Our results have led to valuable insights for enzyme
design guided by conformational dynamics as revealed by
MD computations. Hints of this kind for reshaping the
catalytic pocket for substrate capture, turnover and
stereoselectivity provide an effective strategy for directed
evolution of enzymes in exploring the substrate specificity
and optimizing stereo- and regioselectivity as well as
activity.
(
Figure 4). In the case of the aliphatic ketones, variant T15
shows excellent performance for 3j with reversed (R)-
enantioselectivity (99% ee at full conversion). Aryl
ketones 3l and 3m were reduced with improved
enantioselectivity, and importantly in the case of ketone
3
n, both of the mutants showed full conversion with
reversed excellent enantioselectivity (>99% ee).
Experimental Section
Materials. PrimeSTAR DNA polymerase was obtained
from TAKARA. Restriction enzyme Dpn I was bought
from NEB. The oligonucleotide synthesis and DNA
sequencing were conducted by GENEWIZ technology.
Plasmid preparation kit was ordered from TIANGEN
biotech. All reagents and chemicals were purchased from
commercial sources and used without further purification.
The gene coding TbSADH optimized for expression in E.
coli was cloned into pRSFDuet-1 and constructed by
[
3a]
previous co-worker.
Model generation and substrate docking. All protein
and ligand structures were prepared in the Schrödinger
[
24]
Maestro software. The X-ray structure of ADH from
Thermoanaerobium brockii was used as the basis for
[
7]
model creation. The initial structure of the T15 variant
was generated employing the PyMol program
(
http://www.pymol.org) using the WT model as a template,
and then was performed MD simulations procedures.
Figure 4. Asymmetric transformations of diaryl, aliphatic
and aryl ketones.
Docking ketone 1 to the T15 variant was performed using
[
25]
Glide with standard precision (SP) settings. Induced fit
[
26]
docking (IFD) was also performed, in order to allow the
mutated residues to adapt to the presence of the substrate.
Conclusion
MD simulations. MD simulations were performed with
GPU version of Amber 2016 (comprised of AmberTools16
and Amber16) . Each apo-protein was protonated at pH
Loop motion and flexibility at the binding pocket play
crucial roles in the optimal functions of enzymes.
[
27]
[
8d, 9-14]
7
.4 (to mimic the the experimental conditions) using H++
Induced fluctuation by introducing appropriate mutations
lead to loop movements that can reshape the binding
pocket, and therefore influence substrate capture and
turnover. To exploit this hypothesis in the present study,
the thermostable alcohol dehydrogenase TbSADH was
chosen as the model enzyme in asymmetric reduction of
ketone 1 that is not accepted by the WT. By using the
known X-ray structure of TbSADH, two residues A85 and
I86 were selected for saturation mutagenesis, expecting on
the basis of RMSF analysis that they are most likely to
influence loop movement. After screening only a limited
number of transformants, two variants T15 and T24 were
[
28]
[29]
webserver.
The Amber ff14SB force field
was
employed for the protein in all of the MD simulations, and
[
30]
the TIP3P water model was used to solvate each system,
ensuring a solvent layer of at least 10 Å from any point on
the protein surface. The force field of zinc ion and its
neighboring atoms (cutoff was set as 2.8 Å) were
[
31]
parameterized using ‘MCPB.py’ modeling, using HF/6-
1G* calculations. The carbonyl oxygen atom of ketone 1
3
and other three atoms from residues 37, 59 and 150 were
attached to zinc ion by coordinate bonds. After proper
parameterizations and setup, the resulting system’s
geometries were minimized (5,000 steps for steepest
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Advanced Synthesis & Catalysis
10.1002/adsc.201900249
conjugate and 5,000 steps for conjugate gradient) to
remove poor contacts and relax the system. The systems
were then heated from 0 to 303 K under the constant
amount of substance (N), volume (V) and temperature (T)
one hour with shaking. The crude lysate was centrifuged
for 30 min 4000 rpm at 4 °C. 100 μL of the supernatant
was used for an enzyme activity. A quantity of 250 μL of
supernatant of the active transformants was transferred into
new deep-well plates for reaction with 5 mM ketone 1, 0.1
(
NVT ensemble) for 50 ps. Subsequently, the systems were
+
maintained for 50 ps of density equilibration under
constant amount of substance (N), pressure (P) and
temperature (T) (NPT ensemble) at constant temperature of
mM NADP , and 10% isopropanol for 24 h at 30 °C 800
rpm, the final volume was 400 μL. The remaining substrate
and product were extracted using equal volumes of ethyl
acetate for HPLC analysis by chiral column.
3
03 K (30 °C) and pressure of 1.0 atm using Langevin-
-
1
thermostat (ntt=3) with a collision frequency of 2 ps and
pressure relaxation time of 1 ps. The heating and density
Activity assay. The asymmetric reduction of ketone 1
was conducted by crude enzyme powder of the mutants.
After protein expression of TbSADH mutants induced by
equilibrations were carried out with a weak restraint of 10
kcal mol-1
Å
-2
performed on the protein residues. After
0
.1 mM IPTG, the cells were harvested, washed once with
removal of all restraints applied during heating and density
dynamics, the systems were further equilibrated for 1 ns to
get well settled pressure and temperature for
conformational and chemical analyses. After proper
minimizations and equilibrations, 6 independent 100 ns
MD simulations (with random initial velocities) were
performed for each system. During all MD simulations, the
covalent bonds containing hydrogen were constrained
phosphate buffer (50 mM, pH 7.4), and then lysed by
sonication. The crude extract was obtained by
centrifugation and lyophilized under vacuum to form the
crude enzyme powder which was stored at -20 °C for
further use. The reduction reaction was conducted by
mixing phosphate buffer solution (50 mM, pH 7.4),
+
NADP (1 mM), isopropanol (10% v/v), crude enzyme
powder of the TbSADH mutants (5 mg), and ketone 1 (5
mM) in a round-bottomed flask. The reaction mixture was
stirred at 30 °C, 1000 rpm for 24 h. The product and
remaining substrate were extracted using equal volume of
ethyl acetate for HPLC analysis to measure the conversion
and enantiomeric excess (ee) (Table S4). All the assays
were carried out in triplicate.
[
32]
using SHAKE algorithm, with a MD time-step of 2 fs.
The trajectory file was written every 100 steps. The
binding site volume was calculated by POVME 2.0
software.[ Analysis of trajectories was performed by
33]
CPPTRAJ,[
(
34]
using the hierarchical agglomerative
bottom-up) approach[22] for grouping similar molecular
configurations into distinct sets
Determination
of
kinetic
parameters
and
Site-Directed Mutagenesis. Site-directed Mutagenesis
of TbSADH was constructed using megaprimer
approach[ with PrimeSTAR DNA polymerase. The PCR
conditions for short fragment: 95 °C for 5 min, (95 °C for
thermostability. The TbSADH mutants with His-Tag
were purified by nickel-affinity chromatography and
35]
[4c]
analyzed by SDS-PAGE as previously reported. Protein
concentration was measured using the Bradford method.
Enzyme activity was measured by following the
consumption of NADPH from the decrease in absorbance
3
1
6
0 s, 56 °C for 30 s, 72 °C for 40 s,) ×32 cycles, 72 °C for
0 min. For mega-PCR: 95 °C for 5 min, (95 °C for 30 s,
0 °C for 30 s, 72 °C for 6 min,) ×26 cycles, 72 °C for 10
-1
at 340 nm using a molar absorption coefficient of 6220 M
min. The PCR products were analyzed on agarose gel by
electrophoresis. 0.5 μL Dpn I was added in 10 μL PCR
reaction mixture and the digestion was carried out at 37 °C
for 3 h. After Dpn I digestion, the PCR products were
directly transformed into electro-competent E. coli
BL21(DE3) to create the mutants or the final library for
-1
cm . One unit of enzyme activity was defined as the
amount of enzyme catalyzing the conversion of 1 μmol
NADPH per minute. The kinetic parameters were obtained
by measuring the initial velocities of the enzyme reaction
and curve-fitting according to the Michaelis-Menten
equation. For ketone 1, the activity assay was performed in
a mixture containing a varying concentration of ketone 1
[
36]
Quick Quality Control
and screening. The two sites
were first individually substituted to the other 19 canonical
(
0.01-1 mM) and 0.1 mM NADPH. For NADPH, 1 mM
[
19]
amino acids by the Tang method. The SM library of
TbSADH was created by mixed primers F1/R or F2/R
ketone 1 and NADPH in the range of 0.01-0.1 mM were
used for the enzyme kinetic assays. For thermostability,
assays were conducted in a 0.2 mL volume of phosphate
buffer (50 mM, pH 7.4) containing ketone 1 (1 mM), 2.5%
(
library design in Figure S12 and the primers list in Table
S2). The combinatorial mutagenesis library was created by
mixed primers F3/R (library design in Figure S13 and the
primers list in Table S3).
(
v/v) acetonitrile, NADPH (0.1 mM) and the purified
enzyme. The enzyme was incubated at different
temperatures (45-90 °C) for 15 min, followed by the
standard assay procedure to measure the residual activity.
All the assays were carried out in triplicate.
Screening of mutagenesis library. Colonies were
picked up in deep-well plates containing 300 μL LB
medium with 50 μg/mL kanamycin and cultured overnight
at 37 °C with shaking. An aliquot of 120 μL was
transferred to glycerol stock plate and stored at -80 °C.
Then, 800 μL TB medium with 0.2 mM IPTG and 50
μg/mL kanamycin was added to the culture plates for 12 h
at 30 °C with shaking for protein expression. The cell
pellets were harvested and washed with 400 μL of 50 mM,
pH 7.4 potassium phosphate buffer and centrifuged for 10
min with 4000 rpm at 4 °C. Then, the pellets were
resuspended in 400 μL of the same buffer with 6 U DNase
I and 1 mg/mL lysozyme for breaking the cell at 30 °C for
Substrate scope investigation. The asymmetric
reductions of prochiral ketones were catalyzed by crude
enzyme power of the TbSADH mutants. The reduction
reactions were conducted by mixing phosphate buffer
+
solution (50 mM, pH 7.4), NADP (1 mM), isopropanol
(
(
10% v/v), crude enzyme powder of the TbSADH variants
5 mg), and ketone substrate (10 mM) in a round-bottomed
flask. The reaction mixture was stirred at 30 °C, 1000 rpm
for 24 h. Then the product and remaining substrate were
extracted using an equal volume of ethyl acetate for HPLC
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Advanced Synthesis & Catalysis
10.1002/adsc.201900249
analysis and n-butanol for GC analysis. The conversion
and ee values were determined by chiral HPLC and GC
analyses. The absolute configuration was determined by
comparison of the elution order with literature data for
HPLC analysis and performed with Chiralcel OD-H
column (4.6 mm×250 mm×5 μm) and Chiralpak AD-H
column (4.6 mm×250 mm×5 μm). Some of them were
determined based on authentic racemic mixture and
enantiomer product for GC analysis, and performed with
Hydrodex-β-TBDAc (25×0.25 mm ID). Analytic
conditions of HPLC and GC are listed in Table S4 and
Table S5.
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inoculated into test-tube containing LB (5 mL) and
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7 °C until the OD600 reached 0.8-0.9. IPTG was then added
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+
NADP (0.1 mM), and ketone 1 (100 mM) dissolved in 10%
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8
[
+
2
g of wet cells, 6 g/L of ketone 1, 0.1 mM NADP , and 10%
of isopropanol in a final volume of 20 mL. Bioconversion was
carried out at 30 °C, 220 rpm. Reaction completion was
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CDCl
3
) δ 8.45 (d, J = 4.4 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H),
.27 – 7.17 (m, 4H), 7.15 – 7.08 (m, 1H), 7.05 (d, J = 7.9 Hz,
H), 5.64 (s, 1H), 5.20 (s, 1H). 13C NMR (100 MHz, CDCl
7
1
3
)
δ 159.60 , 146.91 , 140.72 , 135.98 , 132.65 , 127.68 , 127.24 ,
21.59 , 120.49 ,120.29 , 73.21.
1
Acknowledgements
This work was financially supported by CAS Pioneer Hundred
Talents Program (No. 2016-053), National Natural Science
Foundation of China (No. 31700698, 21532008, 21676120, and
2
1772142), the Key Research Program of the Chinese Academy
of Sciences (No. KFZD-SW-212), the Natural Science Foundation
of Tianjin (No. 18JCYBJC24600), Open Project Funding of the
State Key Laboratory of Bioreactor Engineering (No.
2
017OPEN02), the Key Projects in the Tianjin Science &
Technology Pillar Program (No. 15PTCYSY00020), and the
National Basic Research Program of China (973 Program:
2
014CB745100). Beibei Liu is funded by the Priority Academic
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FULL PAPER
Conformational Dynamics-Guided Loop
Engineering of an Alcohol Dehydrogenase:
Capture, Turnover and Enantioselective
Transformation of Difficult-to-Reduce Ketones
Adv. Synth. Catal. Year, Volume, Page – Page
Beibei Liu, Ge Qu, Jun-Kuan Li, Wenchao Fan,
Jun-An Ma, Yan Xu, Yao Nie,* and Zhoutong
Sun*
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