Molecular Basis for the High Activity and Enantioselectivity of the Carbonyl Reductase from Sporobolomyces salmonicolor toward α-Haloacetophenones

Article abstract of DOI:10.1021/acscatal.8b00591

In an effort to develop a practical method for the synthesis of optically pure 2,2,2-trifluoro-1-phenylethanol, we found that the carbonyl reductase (SSCR) from Sporobolomyces salmonicolor showed excellent activity and enantioselectivity toward the halogenated acetophenones. Especially, SSCR exhibited more than 1000 times higher activity toward α,α,α-trifluoroacetophenone than unsubstituted acetophenone, a strikingly different observation from the previously well-studied alcohol dehydrogenase (LBADH) from Lactobacillus brevis. Enzyme-substrate docking and site-directed mutagenesis studies revealed the molecular basis for the high enzyme activity and enantioselectivity of SSCR toward the α-halogenated acetophenones. The hydrogen bond of the Asn207 side chain with the substrate halogen atom and the XH/π interaction of the substrate phenyl group with the side chains of Ser222/Thr223 resulted in the formation of the highly reactive conformation of α-halogenated acetophenones in the active site of the enzyme. (S)-2,2,2-Trifluoro-1-phenylethanol was prepared in excellent isolated yield and enantiomeric excess from the reduction of α,α,α-trifluoroacetophenone with mutant T209A. These results suggest that tuning the interactions between the halogen atoms/phenyl group of the substrate and the amino acid residues of the enzyme would lead to valuable mutants for the practical synthesis of β-haloalcohols.

Full text of DOI:10.1021/acscatal.8b00591

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Molecular Basis for the High Activity and Enantioselectivity of the Carbonyl
Reductase from Sporobolomyces salmonicolor toward #-Haloacetophenones
Xi Chen, Hongliu Zhang, Jinhui Feng, Qiaqing Wu, and Dunming Zhu
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00591 • Publication Date (Web): 21 Mar 2018
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Molecular Basis for the High Activity and
Enantioselectivity of the Carbonyl Reductase
from Sporobolomyces salmonicolor toward
αꢀHaloacetophenones
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Xi Chen†, Hongliu Zhang†, Jinhui Feng, Qiaqing Wu, Dunming Zhu *
National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering
Research Center of Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China
* Email: zhu_dm@tib.cas.cn
ABSTRACT: In an effort to develop a practical method for the synthesis of optically
pure 2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol, we found that the carbonyl reductase (SSCR)
from Sporobolomyces salmonicolor showed excellent activity and enantioselectivity
toward the halogenated acetophenones. Especially, SSCR exhibited more than 1000
times higher activity toward α,α,αꢀtrifluoroacetophenone than unsubstituted
acetophenone, a strikingly different observation from the previously wellꢀstudied
alcohol dehydrogenase (LBADH) from Lactobacillus brevis. Enzymeꢀsubstrate
docking and siteꢀdirected mutagenesis studies revealed the molecular basis for the
high enzyme activity and enantioselectivity of SSCR toward the αꢀhalogenated
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acetophenones. The hydrogen bond of the Asn207 side chain with the substrate
halogen atom and the XH/π interaction of the substrate phenyl group with the side
chains of Ser222/Thr223 resulted in the formation of the highly reactive conformation
of αꢀhalogenated acetophenones in the active site of the enzyme.
(S)ꢀ2,2,2ꢀTrifluoroꢀ1ꢀphenylethanol was prepared in excellent isolated yield and
enantiomeric excess from the reduction of α,α,αꢀtrifluoroacetophenone with mutant
T209A. These results suggest that tuning the interactions between the halogen
atoms/phenyl group of the substrate and the amino acid residues of enzyme would
lead to valuable mutants for the practical synthesis of βꢀhaloalcohols.
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KEYWORDS: αꢀhaloacetophenones, carbonyl reductase, enzymatic reduction,
siteꢀdirected mutagenesis, substrateꢀbinding mechanism
Introduction
Stereoselective reduction of aromatic ketones with αꢀhalo groups is of particular
interest, since the corresponding chiral halohydrins (βꢀhaloalcohols) are
keyꢀintermediates in the synthesis of a number of biologically active compounds.1ꢀ4
Chemical asymmetric reduction of monoꢀ or multiꢀ halomethyl ketones has been
widely studied. For example, chiral oxazaborolidine,5 Ruꢀcomplex,6ꢀ7 Irꢀcomplex,8
Feꢀcomplex,9 Corey–Bakshi–Shibata (CBS) catalyst10 and cinchonineꢀmodified
11
Pt/Al2O3 have been used as the catalysts for the asymmetric reduction of
α,α,αꢀtrifluoroacetophenone, and the enantiomeric excess (ee) value of chiral alcohol
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ranges from 8% to 99%. Recently, carbonyl reductases (alcohol dehydrogenases) have
also been extensively used in the synthesis of chiral alcohols, and proved to be a
powerful tool.12ꢀ15 Carbonyl reductases from Pseudomonas sp.16 Kluyveromyces
thermotolerans,17 Rhodococcus ruber,18 Lactobacillus brevis18 and Scheffersomyces
stipitis CBS 604519 have been used as the biocatalyst for the reduction of
α,α,αꢀtrifluoroacetophenone, and the range of the product ee value is 50 to 99%. The
highest specific activity reported for the reduction of α,α,αꢀtrifluoroacetophenone was
124 U/mg, but the ee value was only 50%.19
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In an effort to develop a practical method for the synthesis of optically pure
2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol, we screened the reductases available in our
laboratory. Fortunately, a carbonyl reductase (SSCR) from Sporobolomyces
salmonicolor,20 which belongs to shortꢀchain alcohol dehydrogenase, showed an
enzyme activity of 31.24 U/mg and enantioselectivity of >98% ee. However, its
specific activity toward acetophenone was low (0.03 U/mg) with the ee of the product
alcohol being 42%.21 It has been reported that the specific activity of the alcohol
dehydrogenase
(LBADH)
from
Lactobacillus
brevis
toward
α,α,αꢀtrifluoroacetophenone was only 20% of that for acetophenone.22 The opposite
observations stimulated our curiosity about how the halogen atoms at the αꢀposition
of acetophenone affect the enzyme activity and enantioselectivity. Therefore,
acetophenones with different kinds and number of halogen atoms were examined with
the enzyme SSCR. The enzymeꢀsubstrate docking and siteꢀdirected mutagenesis
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studies were performed to understand the structural basis for the observed enzyme
activity and enantioselectivity.
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Results and Discussion
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The specific activities of purified SSCR enzyme toward six αꢀhalogenated
acetophenones together with αꢀhydroxyacetophenone (Figure 1) were determined by
measuring the absorbance decrease of NADPH in the reaction system. The ee values
of the product alcohols were measured by chiral HPLC analysis of the product
mixtures. The results are summarized in Table 1. As observed in our previous
studies,21 SSCR showed relatively low specific activity (0.03 U/mg) and
enantioselectivity toward acetophenone (1a). It was surprising that the enzyme
specific activity (31.24 U/mg) toward α,α,αꢀtrifluoroacetophenone (1g) was enhanced
more than 1000 times compared to that with 1a as substrate, and the ee of the product
alcohol was also enhanced from 42% (R, 1ꢀphenylethanol) to 98% (S,
2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol). When the αꢀposition was substituted with halogen
atoms, the enzyme specific activity was enhanced at least 223 times. Especially, for
substrates 1e, 1f and 1g with more than two halogen atoms, the enzyme specific
activity was increased up to 1041 folds. Furthermore, the ee values of the product
alcohols were also significantly improved from 42% (R, 1ꢀphenylethanol) to 87 −
>98%. Rosen et al studied the influence of the CF3 group on the activity and
enantioselectivity of various alcohol dehydrogenases, and found that the CF3 group
exerted negative or no influence on the enzyme activity.23 In contrast, the carbonyl
reductases from Pseudomonas sp. strain PED, Leifsonia sp. S749, Kluyveromyces
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thermotolerans and Scheffersomyces stipitis CBS 6045 showed moderate activity
enhancement (7 to 60 times higher) toward 1g compared to 1a.16, 17, 19, 24 The steric
and electronic effects of different αꢀsubstituted ketones on the specific activity of
LBADH were studied in details.22 LBADH showed higher enzyme activity toward the
acetophenones with one or two hydrogen atoms being substituted with
electronꢀwithdrawing groups such as F, Cl and Br, although the activity enhancement
is not significant as SSCR (Table 1). SSCR showed nearly 10 fold activity
enhancement when the αꢀposition of substrate was substituted by hydroxyl group
(1h),25 while LBADH exhibited the opposite trend. The acetophenones with
monoꢀhalogenation were better substrates of LBADH than those with two halogen
atoms at αꢀposition because of the steric effect. Following this tendency, the activity
of LBADH toward 1g is only 20% of that for 1a.22 This is strikingly different from
the observation for SSCR, which are more than 1000 times more active toward
α,α,αꢀtrifluoroacetophenone than acetophenone.
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Figure 1. Structures of acetophenone derivatives used in this study.
Table 1. Asymmetric reduction of αꢀhaloacetophenones with SSCR
substrate
ee%/conformation Relative activity
of SSCR a
Relative activity
of LBADH b 22
(R group)
by SSCR
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1a (CH3)
1b (CH2Cl)
1c (CH2Br)
1d (CHF2)
1e (CHCl2)
1f (CF2Cl)
1g (CF3)
42 R
98 S
1.0
1.0
23.6
5.5
284.3
247.0
223.7
606.7
510.7
1041.3
9.3
87 S
>98 S
>98 S
>98 S
>98 S
92 S 25
7.8
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4.7
n.d. c
0.2
1h (CH2OH)
0.1
a The relative activity of wt SSCR toward acetophenone (0.03 U/mg) is defined as
1.0.
b The relative activity of LBADH toward acetophenone (kcat/Km 16.1 mMꢀ1·sꢀ1) is
defined as 1.0.
c n.d. means “not determined”.
In order to understand the structural basis of the striking increase in the enzyme
activity and enantioselectivity of SSCR toward α,α,αꢀtrifluoroacetophenone, the
ketones 1a and 1g were docked into the substrateꢀbinding site of SSCR and LBADH,
respectively.
Based
on
the
CDOCKER_ENERGY
and
the
CDOCKER_INTERACTION_ENERGY, two most possible conformations of 1a and
one most possible conformation of 1g in the active site of SSCR were obtained
(Figures 2A and 2B). The conformation of 1a in the active center of LBADH was
obtained directly from the crystal data,26 and docking of 1g into the active site of
LBADH gave one most possible conformation (Figures 2C and 2D).
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Figure 2. Docking of substrate 1a and 1g into the active site of the wt SSCR and
LBADH. A) The two most possible conformations of 1a in SSCR shown in green or
purple, and oriented with the Re or Si face toward the cofactor, respectively. B) The
one most possible conformation of 1g in SSCR shown in green, and oriented with the
Si face toward the cofactor (fluorine atom is shown in yellow). C) The conformation
of 1a LBADH shown in green color and oriented with Re face toward the cofactor. D)
The conformation of 1g in LBADH shown in green color and oriented with Re face
toward the cofactor.
As shown in Figure 2A, the distances between the carbon atom of the carbonyl
group of 1a and C4ꢀH of cofactor NMN in the two conformations were 2.97 and 3.06
Å, respectively. The methyl group of 1a could swing in the opposite direction when
the phenyl group was located at the same position. The little difference of the
distances in the two different conformations of 1a is consistent with the experimental
observation of low stereoselectivity (42% ee) for acetophenone. It is quite different
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that only one most possible conformation of 1g in the active site was obtained, and the
distance of carbon atom of carbonyl group of 1g with C4ꢀH of NMN was 2.76 Å. The
shorter distance suggests higher enzyme activity of SSCR toward 1g, and the higher
frequency of the conformation of 1g leading to the Sꢀenantiomer of the product
alcohol is in line with the experimental stereoselectivity (>98% ee) of SSCR toward
1g. In this reactive conformation, the residue Asn207 of SSCR and the fluorine of 1g
forms hydrogen bond with the distance of fluorine atom of 1g and hydrogen atom of
Asn207 being only 2.35 Å. These docking studies suggest that Asn207 may be
responsible for the high activity and enantioselectivity of SSCR toward 1g. And in
general, the αꢀhalogen atom possibly forms hydrogen bond with hydrogen atom of
Asn207 of SSCR, resulting in higher activity and enantioselectivity toward the
αꢀhalogenated acetophenones. In contrast, the distances of carbon atom of carbonyl
group of 1a and 1g with C4ꢀH of NMN in LBADH were 3.72 and 4.14 Å,
respectively. This is consistent with the observed enzyme activity of LBADH toward
these two substrates. 22
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Figure 3 A) Docking of substrate 1g into the active site of wt SSCR, showing the
amino acid residues of SSCR around 1g. B) Docking of substrate 1g into the active
site of mutant SSCR N207A. C) View of the docking results at the backward direction
of A), only Ser222 and Thr223 were shown in sticks form. D) Docking of substrate 1g
into the active site of mutant SSCR S222A.
From the docking results, it can be seen that residues N207, Y208 and T209 are on
the side of α,α,αꢀtrifluoromethyl group, and N207 forms hydrogen bond with fluorine
atom to anchor the trifluoromethyl group. In addition, S222, T223 and S224 locate on
the side of phenyl group, and at least one of them should have some interaction with
phenyl group. These interactions make the C4ꢀH only at the Si face of carbonyl group
(Figure 3A). In order to clarify the role of the amino acid residues around 1g in
determining the enzyme activity and enantioselectivity toward the αꢀhalogenated
acetophenones, mutations of residues N207, Y208, T209, S222, T223 and S224 to
alanine were conducted. The enzyme activity of six mutants toward αꢀhalogenated
acetophenones were measured, and the results are summarized in Table 2 and 3. As
shown in Table 2, although N207A mutation slightly increased the enzyme activity
toward acetophenone, the enzyme activities of mutant N207A toward the
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αꢀhalogenated acetophenones and αꢀhydroxyacetophenone were significantly reduced.
Furthermore, the mutation of Asn207 only slightly reduced the ee of the product
alcohols in most cases (from >98% to 93% for 1g). On the other hands, mutation of
Y208 and T209 to Ala showed quite different effects on the enzyme activity from
N207A. For mutant Y208A, the specific activity for acetophenone was greatly
reduced, while mutant T209A exerted minimal effect on the activity toward the
unsubstituted acetophenone. Mutation of Y208 and T209 to Ala enhanced the enzyme
activity toward the αꢀhalogenated acetophenones in most cases. Especially, T209A
showed the highest enzyme activity (120.0 U/mg) and >98% ee for substrate 1g.
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Table 2. Asymmetric reduction of αꢀhaloacetophenones with SSCR mutants
WT
N207A
Y208A
T209A
substrate
relative
product relative product relative product relative
product
ee %
(R group)
activity a
ee %
42 R
activity
1.3
ee %
34 R
87 S
68 S
94 S
98 S
97 S
93 S
84 S
activity
0.1
ee %
16 S
activity
0.9
1a (CH3)
1b (CH2Cl)
1c (CH2Br)
1d (CHF2)
1e (CHCl2)
1f (CF2Cl)
1g (CF3)
1.0
50 R
96 S
284.3
247.0
223.7
606.7
510.7
1041.3
9.3
98 S
1.5
2.7
90 S
6.1
87 S
42.3
57.0
57.5
73.6
195.9
1.7
396.7
1536.0
352.5
2603.5
1184.7
0.2
67 S
98 S
>98 S
>98 S
97 S
97 S
513.3
1717.3
648.8
1988.6
4001.1
4.5
93 S
>98 S
>98 S
>98 S
>98 S
92 S
>98 S
>98 S
>98 S
>98 S
88 S
1h (CH2OH)
a The relative activity of wt SSCR toward acetophenone (0.03 U/mg) is defined as
1.0.
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For the mutants S222A, T223A and S224A located at the side of phenyl group, the
enzyme activities toward the αꢀhalogenated substrates were also increased in most
cases, similar to the results observed for mutants Y208A and T209A. These amino
acid residues also exert some effect on the enantioselectivity of SSCR. For mutants
S222A and T223A, the enantioselectivity of the biotransformation was quite different
from the wt SSCR even though the enzyme activity was kept high. When Ser222 was
mutated to Ala, the enantioselectivity was reduced for almost all tested ketones and
the enantiopreference reversal was observed for substrates 1a and 1c.
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Table 3. Asymmetric reduction of αꢀhaloacetophenones with SSCR mutants
WT
S222A
Relative
T223A
Relative
S224A
Relative
substrate
Relative
(R group)
ee %
ee %
ee %
ee %
activity a
activity
activity
activity
1a (CH3)
1b (CH2Cl)
1c (CH2Br)
1d (CHF2)
1e (CHCl2)
1f (CF2Cl)
1g (CF3)
1.0
42 R
98 S
0.3
53 S
47 S
23 R
1.0
42 R
91 S
85 S
99 S
98 S
82 S
81 S
69 S
2.1
18 R
95 S
82 S
98 S
>99 S
98 S
98 S
86 S
283.3
247.0
223.7
606.7
510.7
1041.3
9.3
3.4
14.7
15.3
87 S
420.6
530.8
953.7
448.5
1381.0
3.7
945.1
526.5
605.8
2197.3
976.6
2926.7
5.9
>98 S
>98 S
>98 S
>98 S
92 S
87 S 1535.7
98 S
82 S
783.1
448.3
81 S 1381.5
69 S 3.7
1h (CH2OH)
a The relative activity of wt SSCR toward acetophenone (0.03 U/mg) is defined as
1.0.
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Among the tested mutants, mutant N207A showed obviously reduced enzyme
activity. The kinetic parameters of mutant N207A together with wildꢀtype enzyme
and mutant T209A toward 1g were determined (Table 4). The wt SSCR had the
lowest Km (0.32 mM). The mutant T209A showed higher Km, but the highest kcat (51.3
sꢀ1). Mutant N207A had the smallest kcat and increased Km, resulting in a decrease of
the catalytic efficiency to almost only oneꢀtenth of that of the wildꢀtype enzyme.
These results suggest that residue N207 plays an important role for the high activity of
SSCR toward the αꢀhalogenated acetophenones.
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Table 4. Kinetic Parameters of wt SSCR and mutant enzymes
enzyme
wt SSCR
N207A
T209A
Km (mM)
0.32
kcat (sꢀ1)
20.4
kcat/Km (sꢀ1·mMꢀ1)
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6.6
0.89
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51.3
31.5
In order to understand how the mutant N207A demolished the enzyme activity of
SSCR toward the αꢀhalogenated acetophenones, substrate 1g was docked into the
active site of mutant N207A. By using the standard docking protocol, 1g in the active
center of mutant N207A is shown in Figure 3B. Compared to Figure 3A, in which the
amino acid residue Asn207 wiggles toward 1g and forms a hydrogen bond with the
fluorine atom, the amino acid residue Ala207 in Figure 3B is away from 1g. The
distance of carbon atom of carbonyl group of 1g with C4ꢀH of NMN is increased
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from 2.76 Å to 2.85 Å when Asn207 is mutated to alanine. The demolishment of
hydrogen bond between Asn207 and 1g makes the distance between the carbonyl
group of 1g and C4ꢀH of NMN longer, and should be the reason of the reduced
enzyme activity.
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According to the docking results of 1g into wt SSCR (Figure 3C), we reasoned that
the hydrogen atom at the side chain of Ser222 and Thr223 should form the XH/π
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interactions with the phenyl group, thus reducing the mobility of the phenyl group
and locating the Si face of the carbonyl group toward NMN. When 1g was docked
into mutant S222A, we found that the direction of the carbonyl group of 1g has two
opposite orientations, one was the Si face (same as wt SSCR in Figure 3C), and the
other was the Re face (Figure 3D). In both conformations, the trifluoromethyl group
interacted with Asn207. Due to the smaller methyl side chain in the mutant S222A,
the XH/π interaction of the side chain with phenyl group was interrupted or weaken,
and the rest part of substrate tended to rotate, resulting in the reduced
enantioselectivity.
Therefore, we can deduce that the side chain of Asn207 forms hydrogen bond with
the halogen atom when an αꢀhaloacetophenone as the substrate enters the active
center, anchoring the halomethyl group and making the substrate close to the cofactor.
At the meantime, the side chains of Ser222 and Thr223 form XH/π interaction with
the phenyl group of the substrate, fixing the phenyl group at right position. These
multiple interactions make the substrate oriented in a highly reactive conformation in
the enzyme active center, thus leading to high activity and enantioselectivity.
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Since 2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol and its derivatives are important intermediates
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1,
3ꢀ4
for the synthesis of some pharmaceuticals,
the reduction of
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α,α,αꢀtrifluoroacetophenone was conducted with mutant T209A and the GDH
cofactor regeneration system at preparative scale. The substrate was transformed
completely at 100 g/L concentration in 5 h, and the optically pure product alcohol was
isolated in 95% yield, demonstrating the application potential of the mutant SSCR
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enzymes
in
the
synthesis
of
pharmaceutically
important
2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol.
Conclusion
The enzymatic reduction of αꢀhalogenated acetophenones including
α,α,αꢀtrifluoroacetophenone is of synthetic importance. In this study, a different
pattern of the effects of αꢀhalogenation on the enzyme activity from the previously
wellꢀstudied alcohol dehydrogenase LBADH was observed for SSCR. The
siteꢀdirected mutagenesis studies revealed that the enzyme activities of mutant N207A
toward αꢀhalogenated acetophenones were significantly reduced, suggesting the
important role of this residue in determining the enzyme activity toward
αꢀhaloacetophenones. The enzymeꢀsubstrate docking studies imply that the hydrogen
bond (between the side chain of Asn207 of SSCR and the halogen atom of substrate)
and the XH/π interactions (between the substrate phenyl group and the side chains of
Ser222 and Thr223) may be responsible for the formation of the highly reactive
conformation of αꢀhalogenated acetophenones in the active site of the enzyme. These
results provide the molecular basis for the high activity and enantioselectivity of
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SSCR toward αꢀhaloacetophenones with 1g as an example. One of the mutants,
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T209A with the highest enzyme activity and excellent enantioselectivity toward
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α,α,αꢀtrifluoroacetophenone,
was
applied
in
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preparation
of
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(S)ꢀ2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol with excellent isolated yield and ee. These results
suggest that the hydrogenꢀbonding of the halogen atom and the XH/π interactions of
the substrate phenyl group with amino acid residues are critical in determining the
activity and stereoselectivity for the enzymatic reduction of halogenated
acetophenones, thus providing valuable information to guide the future engineering of
carbonyl reductases for efficient synthesis of βꢀhalogenated alcohols.
Experimental Section
General
Carbonyl reductases SSCR, and glucose dehydrogenase (GDH) were prepared as
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previously described.
The αꢀhalogenated acetophenones and the other ketones
were purchased from commercial sources and the cofactors were obtained from
Roche. The racemic alcohol standard samples were prepared by the reduction of the
corresponding ketones with sodium borohydride. The substrate conversions and
product ee values were determined by HPLC analysis using a Chiralcel ODꢀH column
(Daicel Co., Japan; 4.6 mm x 250 mm). The enzyme activities toward the reduction of
ketones were measured using a SpectraMax M2 microplate reader (Molecular
1
Devices). The H NMR spectra were measured on Brucker Avance 400 using CDCl3
as the solvent. When the reaction was conducted, NADP+ not NADPH was added,
because NADPH could be regenerated by GDH and Dꢀglucose.
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The KODꢀPlusꢀDNA Polymerase was obtained from TOYOBO CO., LTD.
Restriction enzyme Dpn I was bought from Thermo Scientific. The oligonucleotides
were synthesized and DNA sequencing was conducted by BGI. Plasmid mini kit used
for extracting plasmid and PCR extraction kit were bought from Omega Bioꢀtek.
Flexible Docking
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The enzyme structure used as the receptor target in this study was the SSCR protein
structure (PDB ID: 1Y1P) bound to NADPH, 29 and LBADH structure (PDB ID: 1zk4)
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bound to NADPH and acetopehone. Flexible dockings of acetophenone and
α,α,αꢀtrifluoroacetophenone into SSCR and LBADH were performed using flexible
docking protocol and the structures of N207A, S222A mutants were obtained by
using the standard mutation protocol of Discovery Studio V4.1 (Biovia – former
Accelrys, USA). The protein active site was determined from PDB site records, and
the active site was defined by a sphere of 11 Å radius. Other parameters were kept on
default settings in Flexible docking.
Construction of SSCR Mutants
The mutant genes were constructed by using PCR amplification methods with
KODꢀPlusꢀDNA Polymerase as decribed in the manual. Based on the SSCR sequence,
appropriate primers were designed. The PCR reaction conditions were as follows:
94°C 2 min, (95°C 15 sec, 58°C 30 sec, 68°C 8 min) × 20 cycles, 68°C 10 min. The
PCR products were analyzed on agarose gel by electrophoresis and purified using a
Omega Bioꢀtek CycleꢀPure kit. The purified PCR products were digested with DpnI
restriction enzyme at 37 °C for more than 1 h to remove template DNA. After
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digestion, the genes were purified again using CycleꢀPure kit. The purified products
were transformed to commercial E. cloi BL21(DE3) by heat shock. Transformation
mixtures were spread on agar plates containing 100 µg/mL ampicillin and grown
overnight at 37°C. The cultures containing the corresponding mutant genes were
confirmed by DNA sequencing (BGI).
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Purification of wt and Mutant SSCR Proteins
E. coli BL21 (DE3) cells expressing wt SSCR and mutant genes were cultivated in
5 mL LB medium containing 100 µg/mL ampicillin overnight at 37°C. The overnight
culture was inoculated into 600 mL LꢀB medium containing 100 µg/mL ampicillin
and grown at 37°C. The culture was induced by addition of isopropyl
βꢀDꢀ1ꢀthiogalactopyranoside (IPTG) with a final concentration of 0.1 mM when
OD600 reached 0.6ꢀ0.8, and then allowed to grow for additional 16 h at 30°C. After
centrifugation at 6000 × g for 15 min at 4°C, the cells were washed with phosphate
buffer (50 mM, pH 7.5), and resuspended in a phosphate buffer (50 mM pH 7.5)
containing 0.5 M of NaCl. The cells were lysed by high pressure homogenizer and the
supernatant was collected by centrifugation at 8000 × g for 20 min at 4°C. The
supernatant was filtered with 0.22 µm filter. Protein purification was performed using
an AKTA purifier 10 system with UNICORN 5 software (GE Healthcare). The wt and
mutant proteins were purified using affinity chromatography with Ni SepharoseTM
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Fast Flow column (GE, USA). The column was preequilibrated with buffer (50 mM
phosphate buffer, 0.5 M NaCl, pH 7.5). The sample (70 mL) was loaded with a flow
rate of 3.0 mL/min. After washing with 50 ml of the equilibrium buffer, the bounded
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target protein was washed with 50 – 75 mM imidazole solution containing 0.5 M
NaCl and 50 mM sodium phosphate (pH 7.5). The target protein fractions were
collected and desalted through ultrafiltration. The pure enzyme solutions were stored
at ꢀ80°C after adding 10% glycerin and 0.1 mM DTT.
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Measurement of the Activity of wt and Mutant Enzymes and the Product ee
Values
The specific activity of the purified carbonyl reductase SSCR and its mutants
toward the reduction of αꢀsubstituted acetophenones was determined by
spectrophotometrically measuring the oxidation of NADPH at 340 nm (ɛ = 6.22 mMꢀ1
cmꢀ1) in the presence of an excess amount of ketones. The activity was measured at
room temperature in a 96ꢀwell plate, in which each well contained ketone (6.25 mM),
NADPH (0.40 mM) and 10% v/v DMSO in sodium phosphate buffer (100 mM, pH
7.0, 190 µL). The reaction was initiated by the addition of the carbonyl reductase (10
µL solution containing 0.5ꢀ20 µg of enzyme) in 5 min. The specific activity (U/mg)
was defined as the number of micromoles of NADPH converted in 1 min by 1 mg of
enzyme.
Sodium phosphate buffer (1 mL, 100 mM, pH 7.0) containing 10% DMSO (v/v), 10
mM substrate, 30 mM Glucose, 0.5 mM NADP+, 0.5ꢀ2 mg SSCR or its mutant and 4
U GDH was shaken at room temperature. The reaction was stopped after overnight by
addition of equal amount of ethyl acetate. The organic phase was separated and the
solvent was removed. The resulting sample was analyzed by chiral HPLC to
determine the ee value of the product alcohol.
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Measurement of Kinetic Parameters for Substrate 1g
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The kinetic parameters were obtained by measuring the initial velocities of the
enzymatic reaction and curveꢀfitting according to the Michaelis−Menten equation
using GraphPad Prism 5 software (GraphPad Software Inc.). The enzyme activity for
ketone reduction was determined by spectrophotometrically monitoring the
absorbance of NADPH at 340 nm at room temperature. The reaction was carried out
in potassium phosphate buffer (100 mM, pH 7.0) with 0.5 mM NADPH. The
substrate concentration range was between 0.1 and 10 mM.
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Enzymatic Reduction of α,α,αꢀTrifluoroacetophenone
A solution of 5 g α,α,αꢀtrifluroacetophenone in DMSO (5 mL) was added into a
sodium phosphate buffer (100 mM, pH 7.0, 45 mL) containing carbonyl reductase
SSCR T209A 50 U (1 U was defined as the number of micromoles of NADPH
converted in 1 min with 1g as substrate), lyophilized powders of GDH 50 U, NADP+
(5 mg), and 12 g Dꢀglucose. The reaction mixture was stirred at room temperature
with TLC monitoring from time to time. The pH was controlled at 7.0 by adding 2 M
NaOH. After complete consumption of the substrate, the reaction mixture was
extracted three times with ethyl acetate. The combined organic layers were dried over
anhydrous Na2SO4 and evaporated under vacuum to afford the optically pure product,
(S)ꢀ2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol (4.8 g, purity >95%, 95% yield), without further
purification. 1H NMR (600 MHz, CDCl3) δ 5.01 (q, J = 6.9, 12 Hz, 1H), 7.26–7.57 (m,
5H).
Corresponding Author
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* Corresponding author: Dunming Zhu
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Telephone: 86ꢀ22ꢀ84861962
Fax: 86ꢀ22ꢀ84861996
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Eꢀmail: zhu_dm@tib.cas.cn
Author Contributions
†
X. Chen and H. Zhang contributed equally.
Notes
The authors declare no competing financial interest.
Supporting Information Available
1H NMR spectrum of (S)ꢀ2,2,2ꢀtrifluoroꢀ1ꢀphenylethanol. This information is
available free of charge on the ACS Publications website.
ACKNOWLEDGMENT
This work was financially supported by Tianjin Municipal Science and Technology
Commission (15PTGCCX00060 and 15PTCYSY00020) and National Natural
Science Foundation of China (No. 21602246).
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