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in buffer A (20 mM Tris–HCl, 500 mM NaCl and 30 mM imidazole,
pH 7.5) to 100 mg/mL wet cells. After disrupting the cells by ultrasonic
and removing cell debris, the resulting supernatant was loaded onto a
nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Tiandz, Beijing,
China) preequilibrated with buffer A, and followed by elution at
0.4 mL/min with buffer B as the same as buffer A, except for 300 mM im-
idazole. Aliquots of 1 mL eluents only containing the target EH assayed
by SDS-PAGE were pooled, dialyzed against 20 mM phosphate buffer
(pH 7.0), and concentrated using a 10 kDa cut-off ultrafilter membrane
(Millipore, Billerica, MA).
edu/SAVES/). Synchronously, the 3-D structures of four enantiomeric
substrates, (S)- and (R)-5a (and 7a), were constructed and disposed in
minimized energy using the MM2 force field in the ChemBioOffice
2.11. Molecular docking simulation of GmEH3 with enantiomeric 5a or 7a
The mutual action between the modeled 3-D structures of GmEH3
and (S)- or (R)-5a (or 7a) was predicted by MD simulation using the
by the GROMACS 4.5 package to locate the most appropriate binding
sites and steric orientation, that is, a binding state having the lowest
binding free energy (ΔGbinding) [15]. The ΔGbinding value of each docked
complex was calculated by using the molecular mechanics Poisson-
Boltzmann surface area (MM-PBSA) method [16]. Based on the 3-D con-
formations of simulatedly docked EH-epoxide complexes, such as
GmEH3-(S)-5a and -(S)-7a, the through-space distances (dα and dβ)
and the hydrogen bond lengths (l1 and l2) were identified using a
tance between the nucleophilic side-chain oxygen of Asp101 residue in
GmEH3 and Cα or Cβ in the oxirane ring of (S)- or (R)-5a (or 7a), as
well as the l1 or l2 was the hydrogen bond length from the hydroxyl
group of Tyr152 or Tyr234 residue (proton donor) to the oxygen atom
in an oxirane ring.
2.7. Assay of the kinetic parameters of purified GmEH3 towards enantio-
meric 5a or 7a
The initial hydrolytic rates (μmol/min/mg protein) of (S)- and (R)-5a
(or 7a) catalyzed by purified GmEH3 were determined under the EH ac-
tivity assay conditions, except for the concentrations of (S)- and (R)-5a
(or 7a) ranging from 0.5 to 20 mM. Both the Km and Vmax values of
GmEH3 were calculated by non-linear regression analysis using an Ori-
)
of GmEH3 for (S)- and (R)-5a (or 7a) was deduced from its apparent
MW and Vmax, while its catalytic efficiency (kcat/Km) was defined as
the ratio of kcat to Km. All kinetic parameters from three independent
replicates were expressed as the mean standard deviation (SD).
2.8. Scale-up enantioconvergent hydrolysis of rac-5a by E. coli/gmeh3
3. Results and discussion
The regioselective hydrolytic reactions, in four aliquots of 2 mL
100 mM phosphate buffer (pH 7.0) systems containing 200 mg/mL
wet cells of E. coli/gmeh3 and rac-5a at concentrations ranging from 50
to 200 mM, were carried out, respectively, at 25 °C within 8.0 h. Using
the c and eep values as the criteria, the maximum allowable concentra-
tion (MAC) of rac-5a was first confirmed. Subsequently, the scale-up
enantioconvergent hydrolysis of rac-5a at MAC was carried out in the
50 mL phosphate buffer system. During the hydrolytic process, aliquots
of 50 μL reaction samples were drawn periodically, extracted with
950 μL ethyl acetate, and then analyzed by chiral HPLC with a Chiralcel
OD-H column (Daicel, Osaka, Japan) until rac-5a was almost completely
hydrolyzed (c > 99%). In addition, the space-time yieldp (STYp, g/L/h) of
(R)-5b, which was defined as the amount of (R)-5b produced from rac-
5a per unit volume and time, was calculated to evaluate its production
efficiency.
3.1. Excavation of a novel EH based on the computer-aided analysis
One hGmEH, which shared the highest sequence identity of 81.3%
with a characterized PvEH3 [17], was selected. Then, its multiple se-
quence alignment with six known plant EHs (sharing over 55% identity
with hGmEH) was carried out (Fig. 3a). The alignment result indicated
that hGmEH contained the three typical conserved motifs existing in
all α/β-hydrolase fold EHs: HGXP, GXSmXS/T and SmXNuXSmSm, in
which X, Sm and Nu were any, small and nucleophilic residues, respec-
tively [14,18]. It was confirmed that HGXP motif forms an oxyanion hole
to stabilize the negative charge of a nucleophilic side-chain oxygen of
Asp in EH's catalytic triad during the hydrolysis [19]. The catalytic
triad of hGmEH was confirmed as Asp120-His316-Asp281. In addition, its
two proton donors were also conserved as Tyr169 and Tyr251. It was ver-
ified that two specific Tyr residues play important roles in substrate
binding and ring-opening via forming hydrogen bonds with the oxygen
atom in oxirane ring [4,20]. Owing to the above analytic results, it was
speculated that hGmEH may have catalytic activity towards epoxides.
Thus, hGmEH was renamed as GmEH3, and identified as the research
object for the cloning and heterologous expression of a gene coding
for GmEH3, as well as the investigation of its catalytic performances.
Furthermore, the phylogenetic tree analysis on the sequences of
hGmEH and 14 representative EHs revealed that hGmEH (or GmEH3)
was closely related to plant EHs, but relatively distant from those of
other species (Fig. 3b).
2.9. Scale-up kinetic resolution of rac-7a by E. coli/gmeh3
The enantioselective hydrolytic reactions, in six aliquots of 2 mL
100 mM phosphate buffer (pH 7.0) systems consisting of 200 mg/mL
wet cells and rac-7a at elevated concentrations from 100 to 600 mM,
were conducted, respectively, at 25 °C within 8.0 h, and analyzed by chi-
ral HPLC with the Chiralcel OD-H column (Table S2). Using the ees and
yields of (R)-7a as the criteria, the MAC of rac-7a was confirmed. The
gram-scale kinetic resolution of rac-7a at MAC in the 30 mL phosphate
buffer system was conducted until the ees of (R)-7a reached over 99%.
The STYs (g/L/h) of (R)-7a was defined as the amount of (R)-7a retained
from rac-7a per unit volume and time.
3.2. Cloning and intracellular expression of gmeh3
2.10. Homology modeling of GmEH3 and enantiomeric 5a or 7a
An about 1.0-kb nucleotide sequence of gmeh3 was amplified from
G. max total RNA, ligated with pUCm-T, and transformed into E. coli
JM109. DNA sequencing result verified that gmeh3 (GenBank no.
MN833949) was exactly 963 bp in length (excluding Nde I and Xho I re-
striction sites), encoding GmEH3 (GenBank no. QJC19071) with 320
amino acid (aa) residues. The catalytic triad of GmEH3, deduced from
Using a known crystal structure of a Vigna radiata EH (VrEH1, PDB:
5XMD) at 2.00 Å resolution as template, which shares 72.3% primary
structure similarity with GmEH3, the three-dimensional (3-D) structure
of GmEH3 was homologically modeled using MODELLER 9.21 program
chanics optimization using the CHARMM27 force field in the GROMACS
the best geometry quality was selected from all the output ones, and
the sequences of cloned gmeh3, was confirmed as Asp101-His299
-
Asp264, while its proton donors as Tyr152 and Tyr234. Its sequence iden-
tities with hGmEH and six known plant EHs were listed as follows:
hGmEH (XP_006604802, 93.4%), PvEH3 (AKJ75509, 85.3%) [17],