Preparation of structurally diverse chiral alcohols by engineering ketoreductase CgKR1

Article abstract of DOI:10.1021/acscatal.7b01933

Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg^-1 (54-775 U mg^-1). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g^-1 L^-1) were reduced completely in gramscale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g^-1 L^-1 day^-1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a similar active pocket to that in CgKR1.

Full text of DOI:10.1021/acscatal.7b01933

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Article
Preparation of Structurally Diverse Chiral
Alcohols by Engineering Ketoreductase CgKR1
Gao-Wei Zheng, Yuan-Yang Liu, Qi Chen, Lei Huang, Hui-Lei Yu, Wen-
Yong Lou, Chun-Xiu Li, Yun-Peng Bai, Ai-Tao Li, and Jian-He Xu
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01933 • Publication Date (Web): 13 Sep 2017
Downloaded from http://pubs.acs.org on September 15, 2017
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ACS Catalysis
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Preparation of Structurally Diverse Chiral Alcohols by Engineering
Ketoreductase CgKR1
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GaoꢀWei Zheng,*,†,ǁ YuanꢀYang Liu,†,ǁ Qi Chen,†,ǁ Lei Huang,† HuiꢀLei Yu,† WenꢀYong Lou,‡ ChunꢀXiu
Li,† YunꢀPeng Bai,† AiꢀTao Li,§ and JianꢀHe Xu*,†
†State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East
China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
‡Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou
510640, Guangdong, China
§Department of Biocatalysis, MaxꢀPlanckꢀInstitut für Kohlenforschung, KaiserꢀWilhelmꢀPlatz 1, Mülheim an der Ruhr
45470, Germany
ABSTRACT: Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enꢀ
zymes often restricts their use in industrial applications. Based on computational analysis and previous reports, two residues (F92
and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1ꢀ
F92C/F94W variant was obtained that exhibited higher activity towards all 28 structurally diverse substrates examined than the
wildꢀtype enzyme. Among them, 13 substrates have a specific activity over 50 U mg−1 (54 to 775 U mg–1). Using CgKR1ꢀ
F92C/F94W as a catalyst, five substrates at high loading (>100 g–1 L–1) were reduced completely in gramꢀscale preparative reacꢀ
tions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee)
and high spaceꢀtime yield (up to 583 g–1 L–1 d–1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94
in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a
similar active pocket to that in CgKR1.
KEYWORDS: asymmetric reduction, ketoreductase, chiral alcohol, protein engineering, substrate specificity, biocatalysis
made in recent years to generate new enzymes by protein enꢀ
gineering in an attempt to more effectively reduce the desired
carbonyl substrates under the practical process conditions,10
and facilitate their use in largeꢀscale applications for the synꢀ
thesis of optically active alcohols.11
INTRODUCTION
Optically pure alcohols are highly valuable chiral intermediꢀ
ates used in the synthesis of fine chemicals and particularly
pharmaceutical drugs. Asymmetric reduction of carbonyl
compounds by biocatalysis to generate chiral alcohols repreꢀ
sents a more environmentally friendly and sustainable process
than traditional chemical synthesis methods. This approach
operates under milder conditions and can generate products in
100% theoretical yield with exceptionally high stereoselectiviꢀ
ty.1 Owing to its green credentials, considerable effort has
been devoted to developing ketoreductases for the stereoselecꢀ
tive synthesis of chiral alcohols of pharmaceutical interest.2
However, using naturally occurring enzymes as catalysts for
the largeꢀscale manufacture of chiral alcohols can suffer from
intrinsic limitations, such as low catalytic activity (often <10 U
mg–1 protein) towards nonꢀnatural substrates, insufficient
activity at high substrate/product concentrations (>100 g–1 L–1),
and high catalyst loading (often >20 g–1 L–1). Accordingly,
only very few wildꢀtype (WT) enzymes have been reported for
the practical production of chiral alcohols.3,4
Recently, we identified the versatile ketoreductase CgKR1
from Candida glabrata. The enzyme displays broad substrate
specificity but very low catalytic activity towards the majority
of substrates tested (for only one substrate was an activity over
50
U
mg−1
observed).12
A
CgKR1
variant
(F92L/F94V/I99Y/G174A) was constructed by rational design,
and this mutant enzyme exhibited enhanced activity towards
aromatic
αꢀketo
esters
such
as
methyl
orthoꢀ
chlorobenzoylformate (7ꢀfold increase) but significantly
decreased activity towards aliphatic keto esters (24 U mg−1 for
10a compared with 114 U mg−1 for WT CgKR1).13 Very
recently, You and coꢀworkers also successfully engineered WT
CgKR1 by modifying Phe92 and Tyr208 residues, affording
mutants with higher activity and antiꢀPrelog stereoselectivity
towards αꢀhalo ketones.14,15 Although several positive CgKR1
mutants have been identified, these enzymes only display high
specific activity towards a particular subset of substrates, and
further engineering is required in order to obtain highly active
mutants for more structurally diverse substrates. The main
challenge now is to precisely identify the key residues that
have a marked effect on the catalytic activity of CgKR1.
Although process engineering strategies5 can partially reꢀ
lieve these limitations, advances in protein engineering techꢀ
nologies have proven even much more powerful.6 Various
enzymes have been successfully tailored by protein engineerꢀ
ing to accept nonꢀnatural substrates,7 to catalyze novel reacꢀ
tions8 and to expand the range of substrates accepted.9 Regardꢀ
ing ketoreductases in particular, considerable effort has been
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Herein, we identified two residues probably affecting subꢀ In addition, F94 surrounding the binding pocket is also likeꢀ
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strate binding in CgKR1 by molecular dynamics simulation
and our previous work, and subsequently modified them to
generate mutants with higher activity towards structurally diꢀ
verse compounds. To highlight the industrial potential of the
developed mutants, the largeꢀscale synthesis of pharmaceutical
intermediates was also carried out.
ly to be an important residue affecting enzyme activity, based
on our previous work.12,13 Therefore, a saturation mutagenesis
library comprising all 20 amino acids at this position was also
constructed. Interestingly, only F94W variant with an even
larger side chain showed a slight increase in catalytic efficienꢀ
cy (Table 1, entry 7). Furthermore, the combination of F92C
and F94W resulted in a positive synergistic effect on enzyme
activity; the F92C/F94W double variant not only displayed a
further decrease in Km, but also an additional 2ꢀfold enhanceꢀ
ment in kcat compared with the F92C variant, which resulted in
a considerably higher kcat/Km of 556 s–1 mM–1, representing a
348ꢀfold increase over the WT enzyme (Table 1, entry 8).
Elucidating the Mechanism of the Enhanced Activity. To
investigate the influence of these two residues on enzyme acꢀ
tivity, an allꢀatoms MD simulation of the F92L, F92C and
F92C/F94W mutants was performed in complex with substrate
17a. All four systems (CgKR1ꢀWT, CgKR1ꢀF92L, CgKR1ꢀ
F92C, and CgKR1ꢀF92C/F94W) were simulated and a trajecꢀ
tory of about 50 ns for each system was analyzed in terms of
rootꢀmeanꢀsquare deviations (RMSD) and rootꢀmeanꢀsquare
fluctuation (RMSF) for all Cα atoms using the initial structure
as template (Figure S2).
With CgKR1ꢀF92L, CgKR1ꢀF92C and CgKR1ꢀF92C/F94W
mutants, the smaller side chains of L92 and C92 generate a
larger binding pocket and cause a conformational rearrangeꢀ
ment of the catalytic triad and the pyridine ring of the NADPH
cofactor. In particular, the conformation of the side chain of
Tyr175 shifts towards the inner surface of the binding pocket.
Consequently, the substrate is bound more deeply in the pocket
and forms stronger interactions than in the WT enzyme, as
shown in Figure 1B, 1C and 1D.
Compared with CgKR1ꢀF92L variant, the Cys92 in the
CgKR1ꢀF92C and CgKR1ꢀF92C/F94W variants affects the
binding mode to a much greater extent than Leu92, not only
because of the smaller side chain, but also by facilitating a
more complex and stable hydrogen interaction network beꢀ
tween the sulfur atom and other nearby atoms, as shown in
Figure 1 and Table S2. In the CgKR1ꢀF92C/F94W double
mutant shown in Figure 1D, the indole ring of Trp 94 forms a
stable πꢀπ stacking interaction with the imidazole ring of His93
that is not observed in the CgKR1ꢀF92C variant, as shown in
Figure S3. This shift in the side chain of Trp 94 results in a
significant spatial movement of the substrate. Furthermore, in
addition to potentially stronger binding, the carbonyl group of
the substrate faces towards the catalytic Ser134 and Tyr175
residues, and the substrate appears to be stabilized in the bindꢀ
ing pocket in a more productive conformation. Therefore, the
substrate assumes a more ‘activated’ conformation in the
CgKR1ꢀF92C/F94W mutant.
RESULTS AND DISCUSSION
9
Exploring Key Residues of CgKR1 by Molecular Dyꢀ
namics Simulations. To probe the key amino acid residues
that may affect catalytic activity, molecular dynamics (MD)
simulation was performed on WT CgKR1 in complex with the
difficultꢀtoꢀreduce ketone 17a (Figure 1A). Based on the bindꢀ
ing mode shown in Figure 1A, although the substrate is stabiꢀ
lized in the binding pocket, no strong interactions between the
catalytic triad (Ser134, Tyr175 and Lys179) and ketone 17a
are observed throughout the entire simulation. Detailed analyꢀ
sis of the distance between the carbonyl group of the substrate
and the catalytic Ser134 is shown in Figure S1, and the result
demonstrates that the substrate binding position is distant from
the catalytic residues. Furthermore, we found a strong πꢀπ
stacking interaction between the benzene ring of the Phe92
residue and the benzene ring of the catalytic Tyr175 residue,
which forms a spatial barrier that blocks substrate binding with
the catalytic residues, as has been observed previously for esꢀ
terases.16 We speculated that F92 is a key residue affecting the
activity of CgKR1, hence it was chosen for mutagenesis.
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Table 1. Kinetic Parameters of WT CgKR1 and its Variꢀ
ants for Substrate 17a
Entry
Mutant
Km
(mM)
kcat
kcat/Km
Fold
(s–1)
(s–1 mM–1)
changea
6.50 ±
0.33
3.37 ±
0.39
3.02 ±
0.24
1.56 ±
0.15
4.70 ±
0.03
0.52 ±
0.02
4.25 ±
0.15
0.45 ±
0.03
1
2
3
4
5
6
7
8
WT
F92T
10.4 ± 0.1
8.72 ± 0.28
21.2 ± 0.5
41.2 ± 0.9
156 ± 0.3
128 ± 1
1.60
2.59
7.02
26.4
33.2
246
1.0
1.6
4.4
17
F92I
F92M
F92L
21
F92C
154
1.4
348
F94W
F92C/F94W
9.25 ± 0.09
250 ± 4
2.18
556
a Fold change improvement in kcat/Km over the WT enzyme.
Subsequently, a saturation mutagenesis library comprising
all 20 natural amino acids at position 92 was initially conꢀ
structed. We first examined the activities of cellꢀfree extracts
(CFEs) of all mutants toward 17a, and five mutants exhibited
much higher activity than the WT enzyme. These five variants
and the WT enzyme were purified and characterized by measꢀ
uring kinetic parameters (Table 1). Compared with the WT
enzyme, all five variants displayed increased catalytic efficienꢀ
cy (kcat/Km). Among them, variant F92L exhibited the highest
kcat with a slight decrease in Km (Table 1, entry 5). Meanwhile,
variant F92C displayed a decrease in Km by one order of magꢀ
nitude and a 12ꢀfold increase in kcat, leading to a 154ꢀfold imꢀ
provement in catalytic efficiency (Table 1, entry 6). The reꢀ
markable decrease in Km confirms that the F92C variant binds
substrate 17a much more strongly than the WT enzyme.
The Molecular Mechanics Poisson Boltzmann (Generalized
Born) surface area (MMꢀPB(GB)/SA) method was used to
estimate the free binding energy of the substrate in four enꢀ
zyme variants. As shown in Table S3, the binding free energy
of substrate 17a was calculated to be −20.22 ± 3.04 kcal/mol
in CgKR1ꢀWT and −18.78 ± 2.32 kcal/mol in CgKR1ꢀF92L.
Interestingly, these values were increased to −12.9 ± 2.91 and
−17.04 ± 3.63 kcal/mol with CgKR1ꢀF92C and CgKR1ꢀ
F92C/F94W mutants, respectively. Although the binding free
energy calculated for the WT enzyme was larger than for the
enzyme variants, the substrate binds distant from the catalytic
residues in the WT structure, as shown in Figure 1A, conꢀ
sistent with lower catalytic activity. Decomposition analysis of
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Figure 1. Analysis of the substrate binding mode in the four enzyme using MD simulations. (A) CgKR1ꢀWT, (B) CgKR1ꢀF92L, (C)
CgKR1ꢀF92C, (D) CgKR1ꢀF92C/F94W.
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the total binding energy indicated that the van der Waals interꢀ
actions (ꢀEVDW) contribute more to substrate binding than
electrostatic interactions (ꢀEPB,elec). We noticed that although
the calculated values differ from the experimental results, the
overall trend in calculated the binding free energy is consistent
with the experimentally determined Km values. The higher
binding affinity of the substrate in the enzyme mutants was
therefore partially responsible for the improved catalytic activꢀ
ity.
Table 2. Specific Activity of WT CgKR1 and its
F92C/F94W Variant Towards Aliphatic Ketones and Keꢀ
toesters
O
O
O
O
O
O
O
1a
4a
5a
O
2a
3a
O
O
O
O
COOH
O
COOH
O
O
8a
O
9a
7a
O
6a
O
Analysis of the Substrate Specificity of the Most Active
Variant. A panel of carbonyl compounds (1a−28a) with a
broad range of structural features were used to characterize the
substrate specificity of the WT reductase and the F92C/F94W
variant. As shown in Table 2 and Table 3, the WT reductase
exhibited an extremely low activity towards most substrates,
and even no activity at all for some. Of the 28 substrates tested,
only one was turned over with a specific activity of over 50 U
mg−1 protein. By contrast, the F92C/F94W variant was considꢀ
erably more active, and exhibited a specific activity above 50
U mg−1 for 13 substrates. For aliphatic substrates, the
F92C/F94W variant also displayed improved activity of beꢀ
tween 2.7ꢀ and 320ꢀfold (up to 775 U mg−1) compared with
WT CgKR1, which is far superior to the M3 variant developed
in our previous work that showed enhanced activity towards
aromatic αꢀketo esters but drastically lower activity towards
aliphatic substrates (Table 2).13 For the best substrate 10a, the
specific activity was further improved to 292 U mg−1 from 110
U mg−1. Meanwhile for azacyclic and aromatic substrates, the
activity was 10ꢀ to 233ꢀfold higher (up to 696 U mg−1) than
that of the WT reductase (Table 3). Even for the challenging
remote aliphatic ketoesters such as 13a, 14a and 15a,17 the
F92C/F94W variant displayed improved activity. In addition,
with the difficultꢀtoꢀreduce ketones such as 16aꢀ18a10c,18 and
challenging αꢀhalo ketones 22a and 23a,12,19 the F92C/F94W
variant exhibited significantly improved activity (up to 301 U
mg−1 with 17a).
O
O
Cl
O
O
12a
O
O
11a
10a
O
O
O
O
O
Cl
O
O
O
15a
O
13a
14a
Entry
Substrate
Specific activity (U/mg)
Fold
change b
WT
F92C/F94W
1
2
1a
2a
0.09
0.48
0.79
0.84
4.1
NAa
NAa
0.20
22
7.7
47
86
98
43
110
14
ꢀ
3
3a
34
4
4a
93
5
5a
58
6
6a
2.1
0.6
64
7
7a
ꢀ
8
8a
320
35
2.7
23
38
25
122
188
9
9a
775
292
20
10
11
12
13
14
15
10a
11a
12a
13a
14a
15a
110
0.88
0.20
0.98
0.18
0.08
7.6
24
22
15
a NA = no measurable activity. b Fold change improvement in the activity of
the F92C/F94W variant over the WT enzyme.
Most pleasingly, the F92C/F94W variant displayed activity
of more than 100 U mg−1 towards nine substrates with quite
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time yield (STY) of 350 g L−1 d−1 (Table 4, entry 1).
Page 4 of 8
different structures, and therefore represents a promising bioꢀ
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5
6
7
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catalyst for the industrial production of the corresponding chiꢀ
ral alcohols. These results confirmed our hypothesis that F92
and F94 are vital residues for improving the catalytic activity
of CgKR1, and could also be modified to expand the substrate
profile of other ketoreductases, particularly those possessing
an active pocket similar to that in CgKR1, such as Gre2p from
Sporobolomyces salmonicolor.20
Table 4. Application of the F92C/F94W Variant for the
Synthesis of Pharmaceutically Relevant Intermediates
Entry Substrate Substrate Time Conversion
ee
STY
(g–1 L–1
d–1)
/product
loading
(h)
/yield (%)
(%)
(g L−1)
1
2
3
4
5
9a/9b
130.1
164.6
100.0
100.0
100.0
7
6
>99.0/77
>99.0/78
>99.0/96
>99.0/89
>99.0/86
>99.0 (S)
97.4 (R)
>99.0 (S)
96.6 (R)
>99.0 (R)
350
520
583
306
173
Table 3. Specific Activity of WT CgKR1 and its
F92C/F94W Variant Towards Azacyclic and Aromatic
Ketones and Ketoesters
10a/10b
17a/17b
25a/25b
27a/27b
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
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4
7
12
Ethyl (R)ꢀ4ꢀchloroꢀ3ꢀhydroxybutyrate [(R)ꢀCHBE] is a useꢀ
ful chiral precursor in the synthesis of biologically active comꢀ
pounds such as Lꢀcarnitine23 and (R)ꢀ4ꢀaminoꢀ3ꢀ
hydroxybutyric acid.24 Recently, several ketoreductases have
been developed for the asymmetric synthesis of (R)ꢀCHBE.25
However, insufficient activity of these WT enzymes has hinꢀ
dered their practical applications. As ethyl 4ꢀchloroꢀ3ꢀ
oxobutanoate 10a is unstable in aqueous solution, a waꢀ
ter/toluene biphasic system (1:1, v/v) was applied for the bioꢀ
conversion of 10a. The F92C/F94W variant could efficiently
catalyze the asymmetric reduction of 10a at 165 g L−1 loading
in 50% toluene within 6 h, generating (R)ꢀCHBE with 97.4%
ee (Table 4, entry 2). Furthermore, the high STY of 520 g L−1
d−1 demonstrated that this bioreduction holds promise for the
synthesis of (R)ꢀCHBE.
Specific activity (U/mg)
Entry
Substrate
Fold
change a
WT
0.80
1.4
F92C/F94W
8.3
16
17
18
19
20
21
22
23
24
25
26
27
28
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
10
215
43
301
7.5
164
0.15
0.18
0.30
1.04
0.11
6.90
16.1
16.1
24.0
0.3
2.88
8.10
31.8
220
19
The piperidine moiety is prevalent in natural products,
pharmaceutical drugs and biologically active molecules. For
example, (S)ꢀNꢀBocꢀ3ꢀhydroxypiperidine [(S)ꢀNBHP] is a
useful synthon in the synthesis of the drug Imbruvica that has
been approved by the US in 2013 for the treatment of lymꢀ
phoma. Recently, the biocatalytic synthesis of (S)ꢀNBHP by
asymmetric reduction of difficultꢀtoꢀreduce ketone 17a has
been developed.10c,26 In the present study, the F92C/F94W
variant could completely reduce 100 g L−1 of 17a within 4 h
(Table 4, entry 3), affording the corresponding product (S)ꢀ
NBHP with >99% ee in 96% isolated yield, and the STY of
583 g L−1 d−1 that is to our knowledge the highest value reportꢀ
ed to date.
45
106
213
233
101
28
25.0
696
453
122
8
470
20
(R)ꢀMandelic acid is the key chiral intermediate for the synꢀ
thesis of semiꢀsynthetic penicillins, cephalosporins, antiꢀtumor
agents and antiꢀobesity drugs.27 In addition, it is also used as
an important chiral resolving agent.28 Asymmetric reduction of
25a by the F92C/F94W variant proved to be an efficient proꢀ
cess for the synthesis of (R)ꢀmandelic acid; 100 g L−1 of 25a
was converted to (R)ꢀ25a with 96.6% ee and 89% isolated
yield (Table 4, entry 4).
Ethyl (R)ꢀ2ꢀhydroxyꢀ4ꢀphenylbutyrate [(R)ꢀHPBE] is an
important chiral intermediate for the synthesis of angiotensinꢀ
converting enzyme (ACE) inhibitors such as benazepril, cilazꢀ
april, enalapril and ramipril used to treat hypertension and
congestive heart failure. Some efficient enzymatic asymmetric
reduction processes with high substrate loading and STY have
been reported.29 Herein, we also developed a highly efficient
bioreduction process using the F92C/F94W variant as catalyst,
in which (R)ꢀHPBE was prepared with >99% ee and an STY
54
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a
Fold change improvement in the activity of the F92C/F94W variant over the
WT enzyme.
Preparative Bioreduction of Substrates Using the
F92C/F94W Variant. Using the most active F92C/F94W
variant, the feasibility for its use in the synthesis of pharmaꢀ
ceutically relevant chiral alcohols was further evaluated. Five
substrates were chosen for asymmetric reduction on a gram
scale. Lyophilized cells expressing the F92C/F94W variant
were combined with glucose dehydrogenase (BmGDH) from
Bacillus megaterium that was used to regenerate NADPH.
(S)ꢀ3ꢀhydrobutyrate [(S)ꢀHBE] is an important chiral pharꢀ
maceutical building block for the synthesis of pheromones21
and antibiotics.22 Using the F92C/F94W variant, 130.1 g L−1 of
substrate 9a was converted completely within 7 h, affording
the corresponding product (S)ꢀHBE with >99% ee and a spaceꢀ
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ACS Catalysis
of 173 g L−1 d−1 (Table 4, entry 5) without any further optimiꢀ
zation.
Finally, the biotransformation of 17a was scaled up to 100
mL to further confirm the feasibility of the process (Scheme
1). 17a (10 g) was converted completely to (S)ꢀNBHP with
>99% ee within 4 h. After extraction and normal workꢀup,
9.81 g of (S)ꢀ17b was isolated in 98% yield and an STY of 589
g L−1 d−1. This result indicates that the bioreduction is a practiꢀ
cal process for the synthesis of the Imbruvica intermediate (S)ꢀ
NBHP.
cal Co. Ltd. (Beijing, China), Methyl pyruvate (8a) was obꢀ
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3
4
5
6
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tained from TCI Development Co. Ltd. (Shanghai, China). 4'ꢀ
Bromoacetophenone (20a) was obtained from SigmaꢀAldrich
(Shanghai, China). Butyrophenone (21a) was obtained from
Energy Chemical Co. Ltd. (Shanghai, China). Methyl benzoꢀ
ylform (24a) was supplied by Jiangxi Keyuan Biopharm Co.
Ltd. (Jiangxi, China). Ethyl 2ꢀoxoꢀ4ꢀphenylbutyrate (27a) was
purchased from Sinopharm Chemical Reagent Co. Ltd.
(Shanghai, China). Methyl oꢀchloromandelate was synthesized
by ourselves.12 (R)ꢀ9b, (S)ꢀ10b and (S)ꢀ17b were obtained
from J&K Chemical Co. Ltd. (Beijing, China). All the other
chemicals and reagents were purchased from authentic suppliꢀ
ers, at least of reagent grade and used without further purificaꢀ
tion.
Mutagenesis. Siteꢀdirected mutagenesis was carried out by
PCR using mutagenic primers (listed in Table S1) and plasmid
pET28aꢀCgKR1 as template according to the manufacturer’s
instructions of QuickChange (Stratagene). The PCR product (2
ꢁL) digested by DpnI was transformed into 50 ꢁL of Escheꢁ
richia coli DH5α competent cells, and colonies after transforꢀ
mation were incubated for DNA sequencing until all the deꢀ
signed mutants were obtained. Subsequently, the plasmid of
each mutant was extracted and transformed into E. coli BL21
(DE3) for target protein expression.
Protein Expression and Purification. E. coli BL21 (DE3)
cells carrying the recombinant plasmid were cultivated in 3
mL LB medium containing Kanamycin (50 ꢁg mL−1) at 37°C
and 180 rpm for 12 h. The overnight culture was inoculated
into 100 mL LB medium and grown at 37 °C. When the culꢀ
ture’s optical density (OD600 nm) reached 0.6ꢀ0.8, 0.1 mM IPTG
was added to induce the enzyme expression at 16 °C for addiꢀ
tional 24 h. The cells were harvested by centrifugation (12,000
× g, 10 min) at 4 °C, washed with 0.9% NaCl solution and
resuspended in sodium phosphate buffer (100 mM, pH 6.0).
The cells were lysed by ultrasonication in an ice bath and the
supernatant was collected by centrifugation at 28,000 × g for
30 min at 4 °C. The protein was purified using affinity chroꢀ
matography with HisTrap FF crude column (GE, USA). The
sample was loaded onto a standard NiꢀNTA affinity column,
and subsequently eluted with 20ꢀ500 mM imidazole solution in
a flow rate of 2 ml/min. The target protein fractions were idenꢀ
tified by SDSꢀPAGE before gathered. Protein concentration
was measured with the Bradford protocol.
Activity Assay. Enzyme activities of WT CgKR1 and its
mutants were determined by measuring the decrease in the
absorbance of NADPH at 340 nm (ε = 6.22 mM−1 cm−1) using
an UVꢀVis spectrophotometer (Shimadzu, Japan) at 30 °C.
The assay mixture of 1 mL was composed of 962.5 ꢁL potasꢀ
sium phosphate buffer (100 mM, pH 6.0), 10 ꢁL substrates
(200 mM), 17.5 ꢁL NADPH (10 mM) and 10 ꢁL purified proꢀ
tein with an appropriate concentration. One unit of enzyme
activity is defined as the amount of enzyme catalyzing the
oxidation of 1 ꢁmol NADPH per minute. All experiments were
conducted in triplicate.
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Scheme 1. Biosynthesis of the Imbruvica intermediate 17b at
100 mL scale.
CONCLUSIONS
MD simulation and previous literature analyses led to the
identification of two residues, Phe92 and Phe94, that probably
affect the activity of CgKR1. Mutagenesis of F92 and F94
resulted in the CgKR1ꢀF92C/F94W variant that exhibited a
marked improvement in activity towards structurally diverse
substrates. Scaleꢀup experiments confirmed the feasibility of
the CgKR1ꢀF92C/F94W variant for practical applications. In
summary, we successfully engineered a useful and versatile
ketoreductase for the practical synthesis of various chiral
alcohols. Additionally, the findings provide guidance for the
development of other similar ketoreductases and related
enzymes.
EXPERIMENTAL SECTION
Chemicals. 2ꢀPentanone (1a), 2ꢀhexanone (2a), 2ꢀoctanone
(3a), 2,5ꢀhexanedione (5a) and NꢀBocꢀ3ꢀpyrrolidinone (16a)
were purchased from Aladdin Chemicals Co. Ltd. (Shanghai,
China). Acetylacetone (4a), ethyl levulinate (11a), ethyl 4ꢀ
acetylbutyrate (13a), 4ꢀchlorobutyrophenone (23a) and
ethyl benzoylacetate (28a) were obtained from Alfa Aesar
(Tianjing, China). Ethyl 4ꢀchloroacetoacetate (10a) was obꢀ
tained from Adamas Reagent, Ltd. 4ꢀOxodecanoic acid (6a),
5ꢀoxodecanoic acid (7a), ethyl 4ꢀchloroꢀ3ꢀoxobutanoate (12a)
and ethyl 5ꢀoxodecanoate (14a) were provided by Xiamen
Bestally Biotechnology Co., Ltd. (Fujian, China). Ethyl acetoꢀ
acetate (9a) and ethyl benzoylformate (22a) were purchased
from Shanghai Macklin Biochemical Technology Co., Ltd.
(Shanghai, China). Ethyl 8ꢀchloroꢀ6ꢀoxooctanoate (15a) was
obtained from Suzhou Fushilai Medicine & Chemical Co., Ltd.
(Jiangsu, China). NꢀBocꢀ3ꢀpiperidinone (17a) was supplied by
Shanghai Hobor Chemical Co., Ltd. (Shanghai, China). Nꢀ
Bocꢀ4ꢀpiperidinone (18a), acetophenone (19a) and 2ꢀ
chloroacetophenone (22a) were purchased from J&K Chemiꢀ
Determination of Kinetic Parameters. The kinetic paramꢀ
eters were determined by measuring the initial rate of enzyꢀ
matic reaction at different substrate concentrations (0.2ꢀ10 mM)
and 0.175 mM NADPH, and then calculated by nonlinear reꢀ
gression according to the MichaelisꢀMenten equation using
OriginPro software. All experiments were conducted in tripliꢀ
cate.
Molecular Dynamics Simulations. To elucidate the strucꢀ
ture mechanism of the catalytic efficiency increase in different
mutants, allꢀatoms molecular dynamics simulations were perꢀ
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formed for WT CgKR1 and CgKR1ꢀF92L, CgKR1ꢀF92C NMR (400 MHz, CDCl3) δ 4.31–4.11 (m, 3H), 3.69–3.51 (m,
1
2
3
4
5
6
7
8
CgKR1ꢀF92C/F94W mutants. The 3D structure for each variꢀ
ant was constructed via homology modeling method using the
structure PDB ID: 4PVD as a template with about 60% seꢀ
quence similarity. AutoDock Vina was then used to obtain the
starting structures of the wild type and CgKR1 mutants in
complex with the substrate 17a.
The LEaP module in Amber11 was used to add missing atꢀ
oms and hydrogen. All the systems were solvated in an explicit
TIP3p water box and extended with a thickness of at least 10 Å
from the protein on each side of the box to generate the buffer
between the protein and the periodic boundary in all direction.
Sodium ions were added to neutralize each system. The topolꢀ
ogy and coordinate files for each system were generated via
Amber FF99SB force filed and the force filed for the substrate
was constructed using antechamber package and Gaussian 03.
The force field parameters for the substrate were listed in Taꢀ
ble S3. Finally, NAMD was used for running all the simulaꢀ
tions.
In the simulations, the Longꢀrange electrostatic interactions
were calculated by the particleꢀmesh Ewald (PME) method.
The van der Waals (vdW) interactions were treated by smoothꢀ
ly turning off between 10.5 and 12 Å using a switching funcꢀ
tion. Each system was subjected to a four steps’ energy miniꢀ
mization with the steepest descents method. Each step was
relaxed for 5000 steps. After minimization, each the simulation
system was heated gradually from 0 K to 300 K in the NVT
ensemble by Langevin dynamics. The NPT equilibration enꢀ
semble was then applied for the next 2 ns, followed by 50ns
NPT production simulation.
2H), 2.83 (s, 1H), 2.70–2.55 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H).
Synthesis of 17b. 1.00 g substrate 17a was converted comꢀ
pletely within 4 h, generating 0.97 g (S)ꢀ17b as a yellow oil in
96% isolated yield. H NMR (500 MHz, CDCl3). H NMR
(400 MHz, CDCl3) δ 3.75 (dd, J = 12.9, 3.5 Hz, 2H), 3.58 (d, J
= 26.5 Hz, 1H), 3.14–2.91 (m, 2H), 2.73 (s, 1H), 1.87 (s, 1H),
1.78–1.67 (m, 1H), 1.44 (d, J = 14.1 Hz, 11H).
1
1
Synthesis of 25b. 1.00 g substrate 25a was converted comꢀ
pletely within 7 h, generating 0.89 g (R)ꢀ25b as a light yellow
9
1
liquid in 89% isolated yield. H NMR (500 MHz, CDCl3). 1H
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17
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20
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23
24
25
26
27
28
29
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NMR (400 MHz, CDCl3) δ 7.46–7.28 (m, 5H), 5.16 (s, 1H),
4.33–4.11 (m, 2H), 3.44 (s, 1H), 1.23 (t, J = 7.1 Hz, 3H).
Synthesis of 27b. 1.00 g substrate 27a was converted comꢀ
pletely within 12 h, generating 0.87 g (R)ꢀ27b as a colorless
1
liquid in 86% isolated yield. H NMR (500 MHz, CDCl3). 1H
NMR (400 MHz, CDCl3) δ 7.32–7.27 (m, 2H), 7.20 (ddd, J =
7.2, 6.1, 1.8 Hz, 3H), 4.20 (ddd, J = 11.1, 9.2, 5.6 Hz, 3H),
2.86–2.64 (m, 3H), 2.12 (dddd, J = 13.7, 9.6, 7.2, 4.0 Hz, 1H),
1.95 (dddd, J = 13.8, 9.2, 7.8, 5.9 Hz, 1H), 1.29 (t, J = 7.1 Hz,
3H).
Asymmetric Reduction of 17a at 100 mL Scale. 10.0 g keꢀ
tone 17a (50 mmol) was added into 95 mL phosphate buffer
((100 mM, pH 6.0) containing 0.5 g lyophilized cells harboring
CgKR1 F92C/F94W variant, 1.0 g dry CFEs of BmGDH, 7.87
mg NADP+ (0.1 mM), 13.5 g glucose (1.5 equiv.) and 5 mL
EtOH. The bioconversion was performed by magnetic agitaꢀ
tion at 30 °C for 6 h, and the pH was maintained at 6.0 using 2
M Na2CO3. After extraction and normal workꢀup, 9.81 g of
(S)ꢀ17b was isolated in 97% yield.
Trajectories generated from 0 nsꢀ50 ns were gathered as
conformational data for all the systems. Several analysis methꢀ
ods were applied for structural investigation such as Root
Mean Square Deviation (RMSD), Root Mean Square Fluctuaꢀ
tion (RMSF) and binding free energy estimation using the
Molecular Mechanics Poisson Boltzmann (Generalized Born)
surface area (MMꢀPB(GB)/SA) method. All the structural
images were generated using VMD.
Gram Scale Preparation of Chiral Alcohols Using the
Most Active F92C/F94W Variant. Biotransformation was
carried out on 10 ml scale by magnetic agitation at 30 °C. For
substrates 9a, 17a, 25a and 27a, the reaction mixtures consist
of a substrate with different loading, 50 mg lyophilized cells
harboring CgKR1 F92C/F94W variant, 100 mg CFEs of
BmGDH, 0.787 mg NADP+ (0.1 mM), 1.5 equivalents glucose,
9.5 mL phosphate buffer (100 mM, pH 6.0) and 0.5 mL EtOH.
For substrate 10a, the reaction mixtures contain a substrate
with different loading, 50 mg lyophilized cells harboring
CgKR1 F92C/F94W variant, 100 mg CFEs of BmGDH, 0.787
mg NADP+ (0.1 mM), 1.5 equivalents glucose, 5 mL phosꢀ
phate buffer (100 mM, pH 6.0) and 5 mL toluene. The reaction
pH was maintained at 6.0 by adding 2 M Na2CO3 and the reacꢀ
tion was monitored by GC until the substrate was converted
completely. The organic phase was extract with ethyl acetate
(3 × 10 mL), dried using anhydrous Na2SO4 and evaporated
under vacuum to obtained optically pure products.
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
Supplementary tables and figures for primers list, molecular
dynamics simulation, HPLC and GC analytic conditions and
SDSꢀPAGE of pure protein; HPLC and GC chromatograms of
chiral alcohols; 1H NMR profiles with the chiral alcohols proꢀ
duced by the best mutant.
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: gaoweizheng@ecust.edu.cn
* Eꢀmail: jianhexu@ecust.edu.cn
.
.
Author Contributions
ǁThese authors contributed equally to this work (G.W.Z., Y.Y.L.
and Q.C.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was financially supported by the National Natural
Science Foundation of China (No. 21472045, 21536004 &
21776085), the Fundamental Research Funds for the Central
Universities (22A201514043) and Shanghai Pujiang Program
(15PJ1401200).
Synthesis of 9b. 1.30 g substrate 9a was converted completeꢀ
ly within 7 h, generating 1.03 g (S)ꢀ9b as a colorless liquid in
1
77% isolated yield. H NMR (400 MHz, CDCl3) δ 4.25–4.11
(m, 3H), 2.69 (s, 1H), 2.44 (qd, J = 16.5, 6.1 Hz, 2H), 1.32–
1.17 (m, 6H).
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