Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna radiata Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity

Article abstract of DOI:10.1021/acs.jafc.7b03804

To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45 °C and high stability at pH 4.5-7.5 and 55 °C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward styrene oxides 1a-3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25 °C. These afforded (R)-1b with >99% ee_p and 78.7% overall yield after recrystallization and (R)-9a with >99% ee_s, 38.7% overall yield, and 12.7 g/L/h space-time yield.

Full text of DOI:10.1021/acs.jafc.7b03804

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2017, 65, 9861-9870
Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna
radiata Epoxide Hydrolase with High Enantioselectivity or High and
Complementary Regioselectivity
Die Hu,†,‡,# Cunduo Tang,§,# Chuang Li,⊥ Tingting Kan,⊥ Xiaoling Shi,⊥ Lei Feng,† and Minchen Wu*,†
†Wuxi Medical School, ‡School of Food Science and Technology, and ⊥Key Laboratory of Carbohydrate Chemistry & Biotechnology,
Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
§Nanyang Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Henan 473061, China
S
* Supporting Information
ABSTRACT: To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from
Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45
°C and high stability at pH 4.5−7.5 and 55 °C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward
styrene oxides 1a−3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting
phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by
molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by
enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25
°C. These afforded (R)-1b with >99% eep and 78.7% overall yield after recrystallization and (R)-9a with >99% ees, 38.7% overall
yield, and 12.7 g/L/h space-time yield.
KEYWORDS: Vigna radiata, epoxide hydrolase, enantioselectivity, enantioconvergence, styrene oxides, phenyl glycidyl ethers
eep of diols. Recently, limonene EH (LEH) mutants catalyzed
the kinetic resolution of racemic styrene oxides and obviously
increased the eep and yield of product (R)- or (S)-phenyl glycol
compared with wild-type LEH; among them, eep of (R)-phenyl
glycol catalyzed by variant SZ649 increased from 21 to 79%
eep.11,12 Comparatively, the enantioconvergent hydrolysis can
give diols with high eep and 100% theoretical yield.13 The
convergent hydrolysis by a single EH can be considered as an
ideal bioprocess, but only a few EHs had the high and opposite
regioselectivity toward two enantiomers of epoxide.14 For plant
EHs, only a Solanum tuberosum EH (StEH) had the high and
complementary regioselectivity toward (R)- and (S)-m-
chlorostyrene oxides, producing (R)-m-chlorophenyl glycol
with 97% eep and 88% yield.15 Additionally, two recombinant
EHs from Vigna radiata, VrEH1 and VrEH2, were applied to
enantioconvergent hydrolysis of p-nitrostyrene oxide, affording
(R)-diol merely with 70 and 82.4% eep, respectively.16,17 It also
should be stressed that an EH is not likely to function optimally
on all types of epoxides. Moreover, EHs for commercial
application have generally been limited to insufficient stability,
low stereoselectivity, and poor substrate tolerance. Hence, it is
necessary to explore more EHs with expected properties, thus
providing more options for the kinetic resolution and
enantioconvergent hydrolysis of various epoxides.
INTRODUCTION
■
Epoxide hydrolases (EHs, EC 3.3.2.x) can catalyze the kinetic
resolution or enantioconvergent hydrolysis of epoxides,
affording enantiopure epoxides and diols that are versatile
synthons for the synthesis of agrochemicals, pharmaceuticals,
and fine chemicals.1 Examples are as follows: (R)-phenyl glycol
was used as a synthetic precursor for arylalkylamine
calcimimetics (R)-(+)-NPS R-568 and NK-1 receptor antago-
nist,2,3 whereas (R)-p-nitrophenyl glycol, (R)-p-chlorophenyl
glycol, and (R)-o-cresyl glycidyl ether were precursors
separately for β-adrenergic blocker (R)-Nifenalol,4 neuro-
protective agent (R)-Eliprodil or antiviral agent EMI37.1,5,6
and cardiovascular hybrid agent Zatebradine.7 Along with the
green wave of global industries, the transformation by EHs as
an environmentally friendly manner with high stereoselectivity
is deemed to be a very attractive alternative to toxic and
substrate-limited chemocatalysis.8
Many EHs have been explored and characterized from plants,
micro-organisms, and mammals, among which some EH-
encoding genes have been cloned, modified, and heterologously
expressed. The stereochemical outcome of catalytic reaction
was highly dependent on the enantio- and regioselectivity of the
given EH−epoxide pair.1 The ideal kinetic resolution by a
single EH with high enantioselectivity can simultaneously
produce enantiopure epoxide and diol with high yields, close to
the maximum value of 50%.9 The resolution of trans-
methylstyrene oxide by Kau2 afforded (1R,2R)-trans-methyl-
styrene oxide with >99% ees and 48% yield and (1R,2S)-diol
with >99% eep and 45% yield.10 However, the majority of EHs
exhibited poor enantio- or regioselectivity, resulting in much
lower than 50% theoretical yield of chiral epoxides or very low
In this work, the full-length cDNA of vreh3, a gene encoding
EH from V. radiata (VrEH3), was amplified from total RNA by
Received: August 15, 2017
Revised: October 19, 2017
Accepted: October 23, 2017
Published: October 23, 2017
© 2017 American Chemical Society
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Scheme 1. Stereoselective Hydrolysis of Racemic Epoxides 1a−9a by E. coli/vreh3 Whole Cells Expressing reVrEH3
sequence was obtained by overlapping the amplified 3′- and 5′-end
cDNA fragments.
RT-PCR, from which an open reading frame (ORF) was
obtained, and then expressed in E. coli BL21(DE3). The
recombinant (re) VrEH3 was purified, exhibiting the excellent
enzymatic properties. Additionally, the catalytic properties of
reVrEH3 and stereoselectivity toward nine racemic epoxides
(Scheme 1 and Table 2) were investigated. To elucidate the
special enantio- and regioselectivity, the interaction of three-
dimensional structures between VrEH3 and (R)-1a, (S)-1a,
(R)-9a, or (S)-9a was analyzed by molecular docking
simulation. Finally, the gram-scale preparations of (R)-1b and
(R)-9a at high concentration were performed, for the first time,
in the buffer-free water system at room temperature, using E.
coli whole cells expressing reVrEH3. The product (R)-1b with
high eep and yield was prepared by enantioconvergent
hydrolysis of 1a, whereas (R)-9a had high ees and yield by
kinetic resolution of 9a. These excellent properties make the
easily available reVrEH3 a promising candidate for industrial
bioprocesses. Further, VrEH3 with the special stereoselectivity
also provides a valuable model for understanding the catalytic
mechanism of EHs.
According to the full-length cDNA sequence, a pair of specific
primers, VrEH-F1 and VrEH-R1, was designed for amplifying both the
ORF and DNA sequences of the coding region of vreh3. In brief, the
ORF sequence was amplified from the first-strand cDNA using VrEH-
F1 and VrEH-R1, whereas the DNA sequence was obtained from the
genomic DNA using the same primers. The recombinant plasmid
containing the ORF sequence flanked by Nde I and Xho I sites, pUCm-
T-vreh3, was transformed into E. coli JM109 and confirmed by DNA
sequencing.
Expression and Purification of reVrEH3. The gene vreh3
excised from pUCm-T-vreh3 by Nde I and Xho I was inserted into
pET-28a(+) digested using the same enzymes and transformed into E.
coli BL21(DE3), generating a recombinant strain, designated E. coli/
vreh3. After the strain was cultured in LB medium containing 50 μg/
mL kanamycin at 37 °C until OD600 reached 0.6−0.8, 0.05 mM IPTG
was added to induce the expression of reVrEH3 at 35 °C for 10 h. The
induced E. coli/vreh3 cells were collected, suspended in 50 mM
Na2HPO4−NaH2PO4 buffer (pH 7.0) containing 500 mM NaCl, and
disrupted by sonication at 0 °C. The expressed reVrEH3 with a 6× His
tag at its N-terminus was purified by nickel-nitrilotriacetic acid (Ni-
NTA) affinity chromatography.20 SDS-PAGE was used to analyze the
expression and purification of reVrEH3. The protein concentration
was determined using the BCA-200 protein assay kit (Pierce,
Rockford, IL).21
Enzyme Activity Assay. The biocatalysis was carried out in a 1
mL Na2HPO4−NaH2PO4 buffer (50 mM, pH 7.0) system, containing
20 mM racemic 1a and a certain amount of E. coli/vreh3 suspension or
purified reVrEH3 solution. After incubation at 25 °C for 15 min, the
reaction was stopped by adding 3 mL of methanol. The sample was
assayed by high-performance liquid chromatography (HPLC), using a
Waters e2695 apparatus (Waters, Milford, MA) equipped with an
XBridge C18 column and a 2489 UV−vis detector. One unit (U) of
EH activity was defined as the amount of E. coli wet cells or reVrEH3
generating 1 μmol 1b per minute under the above assay conditions.
Enzymatic Properties of Purified reVrEH3. The pH optimum of
reVrEH3 was determined under the standard assay conditions, except
20 mM 1a in 50 mM Na2HPO4−citric acid buffer (pH 5.0−7.5) and
Tris-HCl buffer (8.0−9.0). To estimate the pH stability, aliquots of
reVrEH3 were incubated at 25 °C for 1 h at different pH values
(Na2HPO4−citric acid buffer: pH 3.5−7.5 and Tris-HCl buffer: 8.0−
9.0). Its pH stability in this work was defined as a pH region over
which the residual reVrEH3 activity retained over 85% of its original
activity.
MATERIALS AND METHODS
■
Materials. V. radiata purchased from a local supermarket (Wuxi,
China) was used for total RNA and genomic DNA extraction. E. coli
JM109 and plasmid pUCm-T (Sangon, Shanghai, China) were used
for gene cloning and sequencing, while E. coli BL21(DE3) and pET-
28a(+) (Novagen, Madison, WI) were used for expression of vreh3. All
enzymes for gene manipulation were purchased from TaKaRa (Dalian,
China). Racemic 1a and 7a were products of TCI (Tokyo, Japan), and
2a−6a, 8a, and 9a were synthesized according to the reported
methods.18,19 Enantiomers (S)-1a, (R)-1a, (S)-1b, and (R)-1b were
purchased from Energy (Shanghai, China).
Cloning of the cDNA and DNA Sequences of vreh3. The
multiple sequence alignment of known plant EHs, separately from
Glycine max, V. radiata, Phaseolus vulgaris, Nicotiana benthamiana, and
Solanum tuberosum, was performed using the ClustalW2 (https://
www.ebi.ac.uk/Tools/msa/clustalw2) and ESPript (http://espript.
ibcp.fr/). Based on the two highly conserved peptide segments,
MHV/IAEK/LG and S/TWRHQI/M, near the N-termini of aligned
EHs, the degenerated primers (F1 and F2) were designed. All primers
(except for those provided by kits) were synthesized by Sangon
(Shanghai, China) and are listed in Table S1. For amplifying the 3′-
end cDNA of vreh3, the first-strand cDNA was reversely transcribed
from the V. radiata total RNA using the RNA PCR kit (TaKaRa,
Dalian, China) and then subjected to two runs of PCR (nested PCR).
Similarly, for amplifying the 5′-end cDNA, the first-strand cDNA was
reversely transcribed using the 5′-full RACE kit (TaKaRa) and
subjected to the nested PCR.20 All PCR products were gel-purified and
inserted into pUCm-T for DNA sequencing. The full-length cDNA
The temperature optimum of reVrEH3 was determined, at pH
optimum, at temperatures ranging from 20 to 60 °C. For evaluating
the thermostability, aliquots of reVrEH3 were incubated at various
temperatures (20−60 °C) for 1 h. Its thermostability was defined as
the temperature at or below which the residual reVrEH3 activity was
more than 85%.
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Article
The specific activity (U/mg protein) of 1a catalyzed by reVrEH3
was determined under the standard assay conditions, except with the
concentrations of (R)-1a or (S)-1a ranging from 2.5 to 25 mM. The
kinetic parameters, Km and Vmax, of reVrEH3 were calculated by
nonlinear curve fitting function.
RESULTS AND DISCUSSION
■
Analysis of the Primary Structure of VrEH3. The full-
length cDNA sequence of 1500 bp was obtained by overlapping
Stereoselectivity of reVrEH3. Hydrolytic reactions of racemic
1a−9a were performed as follows: 0.9 mL cell suspension containing
100 mg of E. coli/vreh3 wet cells in Na2HPO4−NaH2PO4 buffer (50
mM, pH 7.0) was separately mixed with 0.1 mL of 1a−9a in methanol,
at a final concentration of 10 or 20 mM (Table 2), and incubated at 25
°C. Aliquots of the 25 μL sample were periodically withdrawn,
extracted with 1 mL of ethyl acetate, and assayed by chiral gas
chromatography (GC) or HPLC (Table S2) to calculate the ees, eep,
and conversion ratio (c).
The enantiomeric ratio (E), (kcatS/KmS)/(kcatR/KmR), represents a
degree of preferential hydrolysis of S- over the R-enantiomer, that is,
enantioselectivity. The E value of reVrEH3 toward epoxide was
calculated as E = ln[(1 − c) × (1 − ees)]/ln[(1 − c) × (1 + ees)].22
The regioselectivity coefficients, αS and αR, were related to attacks at
more hindered benzylic carbons (Cα) of (S)- and (R)-epoxide rings,
respectively, and calculated as eep = αS − αR + [ees × (1 − c) × (1 −
αS − αR)]/c.23 The absolute configurations of enantiomers of 2a−9a
and 2b−9b were established by comparing their retention times with
those reported previously.12,13,24−26
Figure 1. SDS-PAGE analysis of the expressed and purified reVrEH3
(A) and chiral GC assay of racemic 1a and produced (S)- and (R)-1b
by reVrEH3 (B). Lane M: standard proteins. Lane 1: E. coli/pET-28a
whole cells. Lane 2: E. coli/vreh3 whole cells. Lane 3: purified
reVrEH3.
Molecular Docking Simulation. The three-dimensional (3-D)
structure of VrEH3 was modeled by the MODELER 9.11 program
(http://salilab.org/modeller/) based on the crystal structure of StEH
(PDB: 2CJP) and then optimized by the GROMACS 4.5 package
(http://www.gromacs.org/).27 Meanwhile, the 3-D structures of (R)-
1a, (S)-1a, (R)-9a, and (S)-9a having the lowest energy were handled
using the ChembioOffice 2010 package (http://www.cambridgesoft.
com/). The molecular docking of VrEH3 with substrate was simulated
using the AutoDock 4.2 program (http://autodock.scripps.edu/) to
locate the most suitable binding position and orientation. A molecule-
docked complex structure was optimized by the GROMACS 4.5
package. The GRPMOS 96 force field and solutions encapsulated by
1.5 nm SPC/E water were used for molecular dynamics (MD)
simulations. The molecule-docked complex structures being fixed and
unfixed were successively energy minimized by 500 step steepest
descent and 1000 steps conjugate gradient algorithms, respectively.
Subsequently, the energy-minimized structure models being fixed and
unfixed were subjected to a 100 ps and 2 ns MD simulation processes
at a temperature of 300 K. The hydrogen bond length (l1 or l2)
between the hydroxyl group of Tyr150 or Tyr232 and O atom of the
oxirane ring and the distance (dα or dβ) between the O atom of Asp101
and the α-carbon atom (Cα) or β-carbon atom (Cβ) were determined
by PyMol (http://pymol.org/).28 Binding free energy of VrEH3
docking with (R)- or (S)-epoxide, as a correlation to the affinity
between them, was calculated by the molecular mechanics Poisson−
Boltzmann surface area (MM-PBSA) method.29
Gram-Scale Preparation of (R)-1b by Enantioconvergent
Hydrolysis. The enantioconvergent hydrolysis of racemic 1a was
carried out at 25 °C in a 100 mL buffer-free water system containing
1.20 g of 1a, giving a final concentration of 100 mM, and 10 g of E.
coli/vreh3 wet cells. Aliquots of a 25 μL sample were periodically
withdrawn to monitor hydrolytic progress by chiral GC. After 1a was
completely hydrolyzed, the reaction mixture was saturated with NaCl
and extracted with 30 mL ethyl acetate three times. The ethyl acetate
fractions were pooled and evaporated at vacuum, followed by
recrystallization with CHCl3, affording highly enantiopure (R)-1b.
Gram-Scale Preparation of (R)-9a by Kinetic Resolution. The
kinetic resolution of racemic 9a was conducted at 25 °C in a 50 mL
buffer-free water system containing 1.64 g of 9a (200 mM) and 1 g of
E. coli/vreh3 wet cells. The hydrolytic progress was monitored by
chiral HPLC. When the ees of (R)-9a reached >99%, the reaction
mixture was extracted with 15 mL of ethyl acetate three times. The
ethyl acetate fractions were pooled, evaporated at vacuum, and purified
by silica gel column chromatography, affording (R)-9a with high
enantiopurity.
Figure 2. Effects of pH values (A) and temperatures (B) on the
catalytic activity and stability of reVrEH3.
Table 1. Kinetic Parameters of Purified reVrEH3 toward
(R)- and (S)-1a
substrate
Km (mM)
Vmax (U/mg) kcat (s−1
)
kcat/Km (mM−1 s−1
)
(R)-1a
(S)-1a
53.15 2.31
6.91 0.32
13.49 0.57
8.12 0.38
8.14
4.90
0.15
0.71
both the 1292 bp 3′-end and 338 bp 5′-end cDNA fragments,
which were amplified by RT-PCR from V. radiata total RNA. It
contains a 94 bp 5′-untranslated region, a 957 bp ORF
encoding 318 aa VrEH3, a 435 bp 3′-untranslated region and a
14 bp polyA tail. Additionally, the 1178 bp DNA fragment of
vreh3 was PCR-amplified from genomic DNA. Compared with
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Table 2. Catalytic Activity and Stereoselectivity of E. coli/vreh3 Whole Cells toward 1a−5a and 7a−9a
a
kinetic resolution
enantioconvergent hydrolysis
b
c
b
c
substrate concn (mM) activity (U/g wc)
E
αR (%) αS (%) time (h) c (%) ees (%) conf. yield (%) time (h) eep (%) conf. yield (%)
1a
2a
3a
4a
5a
7a
8a
9a
20
10
20
10
20
20
20
20
5.22
1.89
5.67
0.25
3.15
2.77
0.34
4.80
4.4
4.5
2.8
2.6
97.1
84.0
89.6
74.2
95.8
4.6
1.0
1.5
1.5
d
65.1
72.2
67.8
91.6 (R)
86.6 (S)
92.2 (R)
33.4
27.8
31.3
7
7
94.1 (R)
85.2 (R)
88.2 (R)
60.9 (R)
50.3 (R)
4.0 (S)
97.0
93.1
94.1
80.4
75.1
6.8
0.8
13
12
11
15
20
15
1.0
11.0
45.3
7.8
1.3
11.4
7.9
2
67.4
69.7
55.0
>99 (R)
>99 (R)
>99 (R)
26.2
22.5
45.0
5.7
2.5
16
3.2 (S)
48.7
9.3
4.1
0.75
3.4 (S)
a
b
c
d
100% conversion ratio. Configuration. Analytical yield. Not determined.
the full-length cDNA, the coding region DNA has two introns
of 142 and 79 bp (Figure S1). Many valuable enzymes have
been identified and characterized from micro-organisms, among
which some recombinant enzymes have been applied for
biotransformation.30−32 It is also an effective approach to
explore more biocatalysts from various, easily available and
cheap crops.13,33 To date, only a few plant EH genes have been
cloned and expressed;1 the successful cloning of vreh3,
therefore, enriched the biological information on EHs and
their genes.
6.0−7.5, over which the pH optimum was 7.0. It was highly
stable at pH values ranging from 4.5 to 7.5, retaining more than
85% of its original activity (Figure 2A). Additionally, the
temperature optimum of reVrEH3, at pH optimum of 7.0, was
45 °C. After being incubated at 20−60 °C for 1 h, reVrEH3
displayed high stability at 55 °C or below and still retained 63%
activity at 60 °C (Figure 2B), whose thermostability was much
higher than those of the majority of EHs, such as A. usamii EH2
and A. niger M200 EH.20,35 In particular, the optimal
temperature and thermostability of VrEH3 were 15 °C higher
than those of VrEH2 with only one amino acid difference in the
primary structure, whose sequence information was released
later (GenBank accession no. AIJ27456).17
A primary structure of VrEH3 was deduced from ORF of
vreh3 (GenBank accession no. KR013755), sharing 57.8 and
72.4% identities separately with those of StEH (AAA81891)
and VrEH1 (ADP68585).16,34 A BLAST search showed that
VrEH3 shared the highest identity of 91.5% with a hypothetical
EH from P. vulgaris (XP_007147004). The multiple sequence
alignment of six plant EHs displayed that VrEH3 contains three
typical conserved motifs, which are identical to those of an α/β
fold EH family: HGXP, GXSmXS/T, and SmXNuXSmSm,
where X, Sm, and Nu represent any residue, small residue, and
The kinetic parameters of reVrEH3 toward two enantiomers
of 1a were determined and summarized in Table 1. The Km
S
toward (S)-1a (Km ) was 6.91 0.32 mM, which was 7.7-fold
R
lower than Km of 53.15 2.31 mM, indicating that reVrEH3
displayed higher affinity or preferential hydrolysis toward (S)-
S
R
1a. Although kcat of 4.90 s−1 was lower than kcat of 8.14 s−1,
the catalytic efficiency (kcat/Km) toward (S)-1a (0.71 mM−1
s−1) was much higher than that toward (R)-1a (0.15 mM−1
nucleophilic residue, respectively. One catalytic triad (Asp101
−
Asp262−His297) and two proton donors (Tyr150 and Tyr232) in
VrEH3 were predicted (Figure S2).
s−1). The ratio of catalytic efficiencies, (kcatS/Km )/(kcatR/ KmR),
S
was calculated to be 4.7. All of these results indicated that
reVrEH3 possesses a moderate enantioselectivity toward
racemic 1a.
Expression and Purification of reVrEH3. After the
recombinant strain, E. coli/vreh3 or E. coli/pET-28a (a negative
control strain), was induced by 0.05 mM IPTG at 35 °C for 10
h, the EH activity of E. coli/vreh3 whole cells toward 1a was
measured to be 5.22 U/g wet cell (wc), whereas no EH activity
was detected in E. coli/pET-28a whole cells. SDS-PAGE
analysis displayed that the apparent molecular weight of
reVrEH3 fused with an extra 20 aa oligopeptide containing a
6× His tag at its N-terminus was 36.8 kDa, which was
consistent with its theoretical one (36507 Da) (Figure 1A).
reVrEH3 was purified to homogeneity by Ni-NTA affinity
column chromatography. The specific activity of reVrEH3
toward 1a was assayed to be 4.71 U/mg protein, which was
higher than those of EHs from Aspergillus niger M200 (0.64 U/
mg),35 Agrobacterium radiobacter AD1 (1.04 U/mg),36 and S.
tuberosum (2.0 U/mg).15 Interestingly, reVrEH3 exhibited
excellent enantioconvergence toward racemic 1a, affording
(R)-1b with 94.1% eep at 100% conversion (Figure 1B and
Table 2). To the best of our knowledge, the eep value of (R)-1b
from enantioconvergent hydrolysis of 1a by a single reVrEH3
was higher than those by CcEH (90% eep),37 StEH (86% eep),15
Kau2 (77% eep), and Kau8 (71% eep).10
Analysis of Substrate Spectrum of reVrEH3. To explore
the application and catalytic mechanism of reVrEH3 toward
different substrates, six styrene oxides 1a−6a and three phenyl
glycidyl ethers 7a−9a were selected and subjected to the
hydrolytic reactions using E. coli/vreh3 whole cells as the
biocatalyst. The catalytic activities of whole cells toward eight
epoxides were determined to be 0.25 to 5.67 U/g wet cell
(Table 2), but no EH activity was detected toward o-
nitrostyrene oxide 6a, which was identical to mbEHs A and
B isolated from mung bean.13 This phenomenon may be
attributed to the steric hindrance caused by ortho-substitution.
In addition, the hydrolytic progress curves of 1a−5a and 7a−9a
catalyzed by reVrEH3 were determined (Figure 3), revealing its
different catalytic properties toward various epoxide substrates.
Enantioselectivity of reVrEH3 toward 1a−5a and 7a−
9a. Corresponding to the c values of eight epoxides ranging
from 30 to 50%, the E values of reVrEH3 were calculated
(Table 2) based on the ees values of retained enantiomers of
epoxides. In detail, reVrEH3 exhibited no or very low
enantioselectivity or preference toward racemic 4a or 5a (E
value of 1.0 or 1.3), moderate enantioselectivities toward 1a−
3a and 7a−8a (E values from 4.4 to 11.4), as well as the high
enantioselectivity toward 9a (E value of 48.7). The kinetic
resolutions of 1a−3a and 7a−9a were carried out at 25 °C by
Enzymatic Properties of Purified reVrEH3. The
enzymatic properties of purified reVrEH3 in this study were
investigated using styrene oxide 1a as the model substrate. The
reVrEH3 exhibited higher catalytic activity at a pH range of
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Figure 3. Hydrolysis progress curves of 1a−5a and 7a−9a catalyzed by E. coli/vreh3 whole cells. Hydrolysis reactions of racemic 1a−5a and 7a−9a
were performed as follows: 0.9 mL cell suspension containing 100 mg of E. coli/vreh3 wet cells in Na2HPO4−NaH2PO4 buffer (50 mM, pH 7.0) was
separately mixed with 0.1 mL of 1a−5a and 7a−9a in methanol, at a final concentration of 10 or 20 mM and incubated at 25 °C. Aliquots of the 25
▲
μL sample were periodically withdrawn and assayed by chiral GC or HPLC to calculate the concentrations of (S)-epoxide ( ) and (R)-epoxide
◆
○
●
[(R)-1a, 3a−5a are shown in black Δ, (R)-2a in red Δ, and (R)-7a−9a in cyan Δ], ees of epoxide ( ), eep of diol ( ) and c ( ).
E. coli/vreh3 whole cells, affording five (R)-epoxides with ees
values from 86.6 to >99% and yields from 27.4 to 45.0%,
respectively, while giving (S)-2a with an ees of 86.6% and yield
of 27.8%. However, styrene oxides 4a−5a were not suitable for
kinetic resolution by reVrEH3 due to its no or low
enantioselectivity. These results indicated that the positions
and electronic properties of the substituents of epoxides greatly
influence the catalytic activity and stereoselectivity of reVrEH3.
To our knowledge, reVrEH3 displayed the highest enantiose-
lectivity toward 9a for remaining (R)-9a among all known
native EHs, such as those from Trichosporon loubierii (E = 41)38
and A. niger LCP (E = 5),28 indicating that it was an ideal
biocatalyst for the kinetic resolution of racemic 9a.
Regioselectivity of reVrEH3 toward 1a−5a and 7a−9a.
The regioselectivity coefficients, αR (or βR = 1 − αR) and αS (or
βS = 1 − αS), were applied to interpret the enantioconvergent
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Figure 4. Molecular docking simulations between VrEH3 and (R)-1a (A), (S)-1a (B), (R)-9a (C), or (S)-9a (D). Active site residues including a
catalytic triad Asp101−Asp262−His297 and two proton donors Tyr150 and Tyr232 are shown as cyan sticks. The hydrogen bond length (l1 or l2) between
the hydroxyl group of Tyr150 or Tyr232 and O atom of the oxirane ring is shown as a green line. The distance (dα or dβ) between the O atom of Asp101
and Cα or Cβ is shown as a black line.
Table 3. Molecular Docking Simulation Parameters of VrEH3 with (R)-1a, (S)-1a, (R)-9a, and (S)-9a
l1 (Å)
l2 (Å)
dα (Å)
dβ (Å)
ΔGbind (kcal mol−1
)
substrate
R
S
R
S
R
S
R
S
R
S
1a
9a
2.9
3.4
3.2
3.2
3.4
2.8
2.8
2.6
4.4
4.1
2.9
3.1
3.0
3.4
4.0
2.8
−20.8
−6.0
−31.7
−25.3
Figure 5. Regioselectivity of reVrEH3 toward two enantiomers of 1a (A) and 9a (B).
hydrolysis of racemic epoxides.1 Here, αR and αS values of
reVrEH3 toward α-carbon atoms of (R)- and (S)-1a−5a and
7a−9a were calculated (Table 2), respectively, based on the c of
epoxides, ees of retained enantiomers, and eep of produced diols
in the hydrolytic process. As a comparison, the calculated
selectivity coefficients toward 1a (αR = 2.8 and αS = 97.1%)
were very consistent with those (αR = 2.5 and αS = 96.7%)
directly determined using (R)-1a and (S)-1a as the substrates.
The regioselective complementarity, that is, the difference
between αS and αR values, of reVrEH3 toward two enantiomers
of 1a was higher than those of mbEH A (αR = 32 and αS =
83%)13 and StEH (αR = 7 and αS = 98%).15 In addition,
reVrEH3 also possessed the high and complementary
regioselectivity toward (R)- and (S)-2a or 3a (αR = 2.6 or
0.8%, and αS = 84.0 or 89.6%) and the lower complementarity
toward (R)- and (S)-4a (αR = 11.0 and αS = 74.2%) or 5a (αR =
45.3 and αS = 95.8%), but hardly any regioselective
complementarities toward 7a−9a (Table 2). For the two
enantiomers of 2a, the regioselective complementarity of
reVrEH3 was higher than that of VrEH1 (αR = 13 and αS =
83%)16 but close to that of VrEH2 (αR = 1.6 and αS = 87.2%).17
Due to the high and complementary regioselectivity as well
as low enantioselectivity, the enantioconvergent hydrolysis of
1a, 2a, or 3a by E. coli/vreh3 whole cells produced (R)-diol with
94.1, 85.2, or 88.2% eep and 97.0, 93.1, or 94.1% yield at 100%
conversion. In our previous work, the regioselectivity of PvEH1
toward 1a was improved by directed evolution. Owing to the
increases of αS and βR from 91.1 to >99% and 53.3 to 86.4%,
respectively, the eep of product (R)-1b from enantioconvergent
hydrolysis of 1a by PvEH1L105I/M160A/M175I, a three-site mutant,
was obviously increased from 33.6 to 87.8%.14
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Molecular Docking Simulation between VrEH3 and
Substrate. Molecular docking simulation between VrEH3 and
substrate was performed to gain insights into the substrate
binding mode and the origin of different enantio- or
regioselectivity.43 The (R)-1a, (S)-1a, (R)-9a, or (S)-9a was
automatically docked into the SBP of VrEH3 using the
AutoDock 4.2 program. Molecular docking models showed
that both (R)-1a and (S)-1a docked in the much broader
tunnel 1, whereas (R)-9a docked in tunnel 1 and (S)-9a in
tunnel 2 (Figure 4).
As show in Table 3, the hydrogen bond lengths (l1 or l2)
toward both enantiomers of 1a and 9a are in the range of 2.8−
3.4 Å, indicating that the substrate binding and potential
activation by two hydrogen bonds from Tyr150 or Tyr232 are
maintained, which is one requirement needed to be fulfilled for
a smooth oxirane ring-opening reaction.44 As discussed by
Bruice, the catalytic efficiency for substrates is dependent on
how often the nucleophile and electrophile are present in near
attack conformation.45 Here, the distances dα and dβ for (S)-1a
and (R)-1a (dα = 2.9 and dβ = 3.0) were much shorter than
those for (R)-1a and (S)-1a (dα = 4.4 and dβ = 4.0), indicating
that Cα of (S)-1a and Cβ of (R)-1a are nearly attacked by
Asp101, thus racemic 1a could be enantioconvergently hydro-
lyzed by reVrEH3 (Figure 5A). Additionally, (S)-9a has
relatively shorter distances (dα = 3.1 and dβ = 2.8) and lower
ΔGbind (−25.3 kcal mol−1) than those of (R)-9a, indicating that
VrEH3 favors (S)-9a, thus being preferentially hydrolyzed
(Figure 5B). Those analytical results suggested that the SBP
plays a crucial role in the substrate specificity and stereo-
selectivity of VrEH3 by influencing the binding and interaction
between active site residues and the substrate.
Figure 6. Time course curves of the enantioconvergent hydrolysis of
racemic 1a (A) and the kinetic resolution of racemic 9a (B).
Preparation of (R)-1b by Enantioconvergent Hydrol-
ysis Using E. coli/vreh3 Whole Cells. To access the practical
capability of reVrEH3, a gram-scale enantioconvergent
hydrolysis of racemic 1a (1.20 g, 100 mM) was performed
using 10 g of E. coli/vreh3 wet cells in a 100 mL buffer-free
water system. The conversion reached nearly 82% within 8 h,
after which the reaction rate decreased rapidly, which may be
caused by the high Km of residual (R)-1a. After incubation at 25
°C for 22 h, (R)-1b was obtained with 92.5% eep at 100%
conversion (Figure 6A). Then, 1.09 g of (R)-1b was obtained
with eep up to >99% at 78.8% overall yield after simple
recrystallization with CHCl3. Thus far, a few reported EHs were
applied to prepare enantiopure diols at high concentration, due
to enzyme instability, substrate water dissolubility, or product
inhibition during the biotransformation process.1 Chen et al.
used the hydrophilic ionic liquids in a two-phase system,
improving the substrate concentration of racemic 1a only from
20 mM to 35 mM catalyzed by mung bean with a 49.4% yield
of (R)-1b.47 Compared with other EHs in Table 4, the
enantioconvergent hydrolysis of racemic 1a can effectively
performed by E. coli/verh3 whole cells at a concentration as
Homology Modeling of VrEH3 3-D Structure. Based on
the crystal structure of StEH (PDB: 2CJP),27 sharing 57.8%
primary structure identity with VrEH3, the 3-D structure of
VrEH3 was homologically modeled. The VrEH3 3-D structure
can be divided into two parts: a α/β-hydrolase domain that
contains a catalytic triad Asp101−Asp262−His29 and a lid domain
that contains two proton donors Tyr150 and Tyr232 (Figure S3).
Interestingly, it contains a “U” shape substrate binding pocket
(SBP) with the active site residues placed in the center, giving
rise to two narrow tunnels (tunnel 1 and tunnel 2), whose
spatial properties have an effect on the substrate preference.28,39
Tunnel 1 was constituted of W102, I105, V126, P127, L129, T137
A143, M144, I151, V264, S267, L268, and M270, whereas tunnel 2 was
constituted of F33, P34, I176, T179, K181, G183, P185, N197, M263
,
,
and F298 (Figure S3). Both tunnels of VrEH3 SBP are
interlinked with each other and also lead eventually to the
exterior of the protein, which dramatically differs with those
SBPs of other α/β hydrolase fold EHs, such as StEH (PDB:
2CJP),27 AnEH LCP (PDB: 3G0I),40 BmEH (PDB: 4G02),41
and MtEHB (PDB: 2ZJF),42 whose tunnels closed at the
internal region.
Table 4. Preparation of (R)-1b by Enantioconvergent Hydrolysis of Racemic 1a Using Several EHs
enzyme source
catalytic form
cells
reaction system
substrate concn (mM) temp (°C) time (h) c (%)
eep (%)
yield (%)
ref
C. crescentus
S. tuberosum
Kau2
phosphate buffer
phosphate buffer
Tris−HCl buffer
n-hexane/ionic liquid
buffer-free water
10
4
25
28
28
35
25
2
93
100
100
90 (R)
86 (R)
77 (R)
95 (R)
92.5 (R)
a
37
15
10
47
b
re-enzyme
re-enzyme
3.5
mung bean
V. radiate
enzyme powder
30
49.4
c
E. coli cells
100
22
100
96.2
this study
a
b
c
No information. Recombinant enzyme. Analytical yield.
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Table 5. Preparation of (R)-9a by Kinetic Resolution of Racemic 9a Using Several Native EHs
enzyme source
catalytic form
cells
reaction system
concn (mM)
temp (°C)
time (h)
ees (%)
yield (%)
40
STY (g/L/h)
0.49
ref
T. loubierii
phosphate buffer
phosphate buffer
culture medium
phosphate buffer
deionized water
45
10
30
25
30
30
25
6
a
>99 (R)
− (R)
>99 (S)
>98 (S)
>99 (R)
38
b
A. niger LCP
B. alcalophilus
B. megaterium
V. radiate
re-enzyme
28
growing cells
re-enzyme
6
24
38
0.015
12.7
24
183
200
32
46
E. coli cells
1
38.7
this study
a
b
No information. Recombinant enzyme.
high as 100 mM in a single buffer-free water system, which has
easy operation, is more environmentally friendly, and benefits
the postprocessing. Considering the high concentration, eep,
and yield of (R)-1b, the applicability of this enantioconvergent
process is also markedly superior to those of other reported
EHs. (R)-Phenyl glycol 1b: white solid; 1.09 g; [α]D20 −41.2 (c
0.50, ethanol), >99% ees; 1H NMR (400 MHz, CDCl3, TMS) δ
7.36−7.26 (m, 5H), 4.95 (dd, J1 = 8.0 Hz, J2 = 3.6 Hz, 1H),
3.85 (dd, J1 = 11.2, J2 = 3.6 Hz, 1H), 3.64 (dd, J1 = 11.2 Hz, J2 =
8.0 Hz, 1H).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.7b03804.
Primers for PCR amplification in this study (Table S1);
chiral GC and HPLC conditions for analysis of epoxides
and diols (Table S2); nucleotide sequence of vreh3 and
its deduced amino acid sequence of VrEH3 (Figure S1);
multiple alignment of six plant EH primary structures
(Figure S2); 3-D structure of VrEH3 (A) and the surface
view of its substrate binding pocket (B) (Figure S3);
chiral GC/HPLC spectra used to determine the
configuration of epoxides 1a−9a and diols 1b−9b
(Figures S4−S6) (PDF)
Preparation of (R)-9a by Kinetic Resolution Using E.
coli/vreh3 Whole Cells. A gram-scale kinetic resolution of
racemic 9a (1.64 g, 200 mM) was performed using 1 g of E.
coli/vreh3 wet cells in a 50 mL buffer-free water system. (R)-9a
with >99% ees and 44% yield and product (S)-9b with 76.2%
eep and 42% yield were obtained after incubation for 1 h at 25
°C (Figure 6B). After being purified by silica gel column
chromatography, 0.64 g of (R)-9a was obtained in >99% ees at
38.7% overall yield, as calculated on the basis of the racemic 9a,
which accounts for up to 77.4% of the theoretical yield. To our
knowledge, some reported EHs favor (R)-9a possessing high
enantioselectivity, such as those from Bacillus alcalophilus24 and
B. megaterium,46 and only a few EHs could preferentially
hydrolyze (S)-9a while retaining the useful (R)-9a (Table 5). In
addition, although Tsukamurella paurometabola EH exhibited
excellent activity and enantioselectivity (E = 58) toward 7a, no
activity was detected for 9a.48 In our work, the kinetic
resolution showed the highest substrate concentration and
space-time yield (200 mM, 12.7 g/L/h) for the biocatalytic
synthesis of (R)-9a catalyzed by reported EHs, which were 4.5-
and 25.9-fold higher than those (45 mM, 0.49 g/L/h) achieved
with T. loubierii.38 Further studies on optimizing reVrEH3
expression and biotransformation processes are in progress to
enhance the utility of reVrEH3 for industrial application. (R)-o-
Cresyl glycidyl ether 9a: colorless oil; 0.64 g; [α]D20 −13.56 (c
0.50, methanol), >99% ees; 1H NMR (400 MHz, CDCl3, TMS)
δ 7.13−7.16 (m, 2H), 6.9 (t, J = 7.6 Hz, 1H), 6.8 (d, J = 8.0 Hz,
1H), 4.25 (dd, J1 = 3.2 Hz, J2 = 11.2 Hz, 1H), 4.00 (q, J = 5.2
Hz, 1H), 3.36−3.40 (m, 1H), 2.92 (t, J = 4.4 Hz, 1H), 2.8 (dd,
J1 = 2.8 Hz, J2 = 4.8 Hz, 1H), 2.25 (s, 3H).
In conclusion, a new plant EH gene from V. radiate was
successfully cloned and expressed in E. coli BL21(DE3). The
reVrEH3 displayed high specific activity, excellent thermo-
stability, and different stereoselectivity toward a broad range of
epoxides. Interestingly, reVrEH3 exhibited very high and
complementary regioselectivity and low enantioselectivity
toward 1a−3a, which is suitable for their enantioconvergent
hydrolysis. Meanwhile, it also had high enantioselectivity but no
complementary regioselectivity toward 9a, which is suitable for
its kinetic resolution. Gram-scale preparations of (R)-1b and
(R)-9a with high enantiopurity (>99 ee), tolerance to high
substrate concentration, and high yield make reVrEH3 an
attractive biocatalyst for industrial bioprocesses.
AUTHOR INFORMATION
■
Corresponding Author
*Fax: +86 510 85327332. E-mail: biowmc@126.com.
ORCID
Minchen Wu: 0000-0003-2833-8436
Author Contributions
#D.H. and C.T. contributed equally to this work as a first
author.
Funding
This work was financially supported by the National Natural
Science Foundation of China (21676117), the Postgraduate
Innovation Training Project of Jiangsu, China (KYLX16_0804
and SJLX16_0472).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Prof. Xianzhang Wu (School of
Biotechnology, Jiangnan University) for providing technical
assistance.
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