Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Article abstract of DOI:10.1073/pnas.1404915111

Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates. epoxide hydrolase X-ray crystallography protein engineering product release bulky substrate We are grateful for access to beamline BL17U1 at Shanghai Synchrotron Radiation Facility and thank the beamline staff for technical support. We also thank Dr. Peter K. Park and Profs. Zhihong Guo and Ran Hong for helpful discussions. This work was supported by National Program on Key Basic Research of China Grant 2011CB710800 (to J.-H.X. and J.Z.), National Grand Project for Medicine Innovation Grant 2012ZX10002006 (to J.Z.), National Natural Science Foundation of China Grant 21276082 (to J.-H.X.), and a grant from the Open Fund from the State Key Laboratory of Bioreactor Engineering (to J.Z.).

Full text of DOI:10.1073/pnas.1404915111

Engineering of an epoxide hydrolase for efficient
bioresolution of bulky pharmaco substrates
Xu-Dong Konga,b, Shuguang Yuanb, Lin Lic, She Chenc, Jian-He Xua,1, and Jiahai Zhoub,1
aState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; bState Key Laboratory of
Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China; and
cNational Institute of Biological Sciences, Beijing 102206, China
Edited by James A. Wells, University of California, San Francisco, CA, and approved September 30, 2014 (received for review March 17, 2014)
Optically pure epoxides are essential chiral precursors for the
production of (S)-propranolol, (S)-alprenolol, and other β-adrener-
gic receptor blocking drugs. Although the enzymatic production of
these bulky epoxides has proven difficult, here we report a method
to effectively improve the activity of BmEH, an epoxide hydrolase
from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl
ether, the precursor of (S)-propranolol, by eliminating the steric
hindrance near the potential product-release site. Using X-ray crys-
tallography, mass spectrum, and molecular dynamics calculations,
we have identified an active tunnel for substrate access and prod-
uct release of this enzyme. The crystal structures revealed that
there is an independent product-release site in BmEH that was
not included in other reported epoxide hydrolase structures. By
alanine scanning, two mutants, F128A and M145A, targeted to
expand the potential product-release site displayed 42 and 25
times higher activities toward α-naphthyl glycidyl ether than the
wild-type enzyme, respectively. These results show great promise
for structure-based rational design in improving the catalytic effi-
ciency of industrial enzymes for bulky substrates.
In this work, we select BmEH, an EH cloned from Bacillus
megaterium ECU1001, to expand its substrate scope for bulky
pharmaco substrate α-naphthyl glycidyl ether (NGE). This en-
zyme is a potential industrial biocatalyst because it has unusual
(R)-enantioselectivity and resolves ortho-substituted PGEs and
para-nitrostyrene oxide with excellent enantiomeric ratios (E > 200)
(22). We first identified the active tunnel of BmEH by solving its
crystal structure complexed with a substrate analog phenox-
yacetamide (POA) and analyzing the routes of substrate entry
and product release by mass spectrum analysis. Alanine scanning
experiments targeted to the potential product-release site of
BmEH resulted in two variants, F128A and M145A, with efficient
bioresolution abilities on NGE. Further kinetic measurements and
structural analysis showed that M145A has much higher activity for
the transition state intermediate formation, whereas both mutants
exhibited expanded product-release site. The M145A BmEH var-
iant has been successfully applied for the preparation of (S)-pro-
pranolol on a gram scale. The engineering of the potential product-
release site described herein should have great promise for struc-
ture-based rational design of better industrial enzymes.
epoxide hydrolase X-ray crystallography protein engineering
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product release bulky substrate
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Results
Structures of BmEH and the BmEH–POA Complex. The overall struc-
ture of BmEH, which is similar to the structures of other
α/β-hydrolases, consists of a catalytic α/β domain and a lid domain
that caps the active site (SI Appendix, Fig. S1). Key residues in-
volved in the catalytic triad (Asp-97, -239, and His-267), binding
motif (Tyr-144 and -203), and oxyanion hole (Phe-30 and Trp-98)
ptically pure epoxides and the corresponding vicinal diols
are valuable chiral building blocks for the production of
O
pharmaceutically active compounds and other fine chemicals (1).
Existing approaches for preparing enantiopure epoxides and
diols include the asymmetric epoxidation or dihydroxylation of
olefin substrates and the resolution of racemic epoxides. These
reactions can be accomplished with either chemical catalysts such
as chiral salen cobalt complexes and porphyrin manganese adducts
or biocatalysts such as monooxygenases and epoxide hydrolases
(EHs) (2–4). In the past two decades, EHs have received much
attention because they are cofactor-independent enzymes that are
“easy to use” for catalyzing the hydrolysis of racemic epoxides to
yield highly enantiopure epoxides and vicinal diols (1, 5, 6).
However, application of EHs in laboratory and industry was often
hindered by their narrow substrate scope, low enantioselectivity,
and regioselectivity, or product inhibition (7, 8).
Significance
Application of epoxide hydrolases in synthesizing chiral drug
compounds has been hindered by their limited substrate range.
The enzymatic production of bulky epoxides has proven re-
markably challenging. In this work, we identified an active tunnel
for substrate access and product release of an epoxide hydrolase
with unusual (R)-enantioselectivity. Mutagenesis targeted to
unblock the steric hindrance in the active pocket or the potential
product release site resulted in variants with much higher activity
toward α-naphthyl glycidyl ether, the precursor of β-adrenergic
receptor blocking drug (S)-propranolol. The strategy presented
here may be a useful alternative choice for rational design of
enzymes toward bulky substrates.
Many protein-engineering efforts have been made to over-
come these drawbacks (9, 10). For example, directed evolution
by error-prone PCR or DNA shuffling has been used to enhance
the activity and enantioselectivity of EHs (11–13). Structure-
guided mutagenesis also generated a few EH variants with im-
proved catalytic performance (14–16). The strategy of iterative
Combinatorial Active Site-Saturation Test (CAST) combines the
rational approach and directed evolution to yield high-quality
and small focused mutant libraries for screening EHs with better
enantioselectivity (7, 17). By mutating residues at the substrate-
binding site, the substrates of EHs have been expanded to in-
clude cyclic meso-epoxides, phenyl glycidyl ether (PGE) deriva-
tives, and other styrene oxide-like analogs (18, 19). However, the
catalytic efficiency of EH is still not satisfactory for bulky epoxide
substrates including precursors of (S)-propranolol, (S)-alpreno-
lol, and other β-adrenergic receptor blocking drugs (20, 21).
Author contributions: X.-D.K., L.L., S.C., J.-H.X., and J.Z. designed research; X.-D.K., S.Y.,
and L.L. performed research; X.-D.K., S.Y., L.L., S.C., J.-H.X., and J.Z. analyzed data; and
X.-D.K., J.-H.X., and J.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The crystallography, atomic coordinates, and structure factors have
been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4G00, 4G02,
4INZ, and 4IO0).
1To whom correspondence may be addressed. Email: jiahai@mail.sioc.ac.cn or jianhexu@
ecust.edu.cn.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1404915111/-/DCSupplemental.
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S6). The T241R/L168E BmEH variant was constructed by in-
creasing the steric hindrance between zones 1 and 2. The cata-
lytically dead mutation H267F was introduced into T241R/L168E
and M145F to prevent the covalent transition state intermediates
from being hydrolyzed. After the BmEH variants were incubated
with the bulky substrate NGE for 10 min, the reaction mixture
was separated by a chromatographic capillary column and further
subjected to mass spectrum analysis. As shown in Fig. 2, in-
cubation of H267F or M145F/H267F with NGE generated a peak
with molecular mass increase of 200 Da, which exactly matches
the theoretical size of NGE. However, we did not detect any
corresponding peaks in the reaction mixture of T241R/L168E/
H167F and NGE, suggesting that the tunnel between zones 1 and
2 plays an essential role in substrate access.
Mutations Expanded the Product Release Site of BmEH. Although the
tunnel between zones 2 and 3 disfavors the substrate access, it is
important for BmEH. As mentioned earlier, blocking this area by
mutating Met-145 to Phe resulted in an ∼80% activity decrease
for PGE and NGE (SI Appendix, Table S2). It is reasonable to
propose that this area may be responsible for product release,
and blocking the product exit caused severe activity loss. Another
possibility is that mutation in this area might induce conforma-
tional changes and thereafter affect the catalysis in zone 2. This
finding offers prospects for generating and screening BmEH
variants with better bioresolution efficiency toward bulky epox-
ide substrates. Among all constructed variants, the activities of
L132A, M145A, and F128A were 13, 25, and 42 times higher
toward NGE, respectively. The kinetic data of M145A and
F128A showed that the enhanced activities were mainly due to
their higher kcat values (57 and 32 times the kcat of wild-type;
Table 1). As a control, mutations of the residues in zones 1 and 2
(L168A, L206A, L219A, F220A, and F242A) did not yield
enzymes with increased catalytic activity toward NGE.
Fig. 1. Three POA binding sites found in the active tunnel of the BmEH–
POA complex. (A) POA molecules in zones 1 (yellow), 2 (green), and 3 (cyan).
(B–D) The POA binding pattern in zones 1 (B), 2 (C), and 3 (D). The residues of
BmEH interacting directly with POA are shown as ball-and-stick models.
are highly conserved in BmEH, HssEH (23), MmsEH (24), MtEH
(25), AnEH (26), ArEH (27), and StEH (28) (SI Appendix, Table
S1). In the BmEH–POA complex structure, three POA molecules
were observed in the catalytic zones (Fig. 1A and SI Appendix,
Fig. S2). One POA molecule (Fig. 1B) was located in a deep cleft
(zone 1) surrounded by residues Leu-168, Tyr-178, Pro-240, Thr-
241, Ser-266, and His-267. Another POA molecule (Fig. 1C) was
deeply buried in the catalytic cavity (zone 2), which was identified
on the basis of structural similarity with the catalytic centers of
other EHs. The amide nitrogen of this POA was hydrogen-
bonded to the side chain of Asp-97 (N–O distance 2.7 Å), which is
responsible for the nucleophilic attack on epoxide in the catalytic
mechanism, and the amide oxygen and the ether oxygen were
hydrogen-bonded to binding motif residues Tyr-144 and -203,
respectively. The substrate analog may have been further stabi-
lized by π–π stacking of the phenyl rings of POA and Trp-98. The
third POA (Fig. 1D, cyan) was located on a bowl-like surface
(zone 3) underneath zone 2; Met-145, Phe-128, Ile-208, and Ser-
142 made up the base of the bowl, and Gln-139, Leu-132, Phe-
209, Lys-146, and Leu-214 made up the side walls.
To interpret the kinetic aspects of F128A and M145A, the
stopped-flow method was used to examine the fluorescence signal
change during the hydrolysis reaction of NGE. Unfortunately,
because of the low signal/noise level, no significant burst-phase
Identification of the Active Tunnel of BmEH. Comparison of the
BmEH–POA complex structure with those of other EHs showed
that there is an active tunnel in BmEH consisting of zones 1, 2,
and 3. Zone 2 is conserved in all EH structures and serves as the
catalytic center. It is accessible from zone 1 and very close to, but
detached from, zone 3 (SI Appendix, Fig. S3). Analysis of the B
factor for each zone in BmEH revealed that the atomic fluctu-
ations of residues 125–156 and 204–225, located between zones 2
and 3, were significant (SI Appendix, Fig. S4). This fluctuation
was confirmed by root mean square (rms) fluctuation and rms
deviation (rmsd) statistics from an 80-ns molecular dynamics
(MD) simulation (SI Appendix, Fig. S5). These results suggested
that such fluctuations might promote the transfer of substrate or
product between zones 2 and 3. Indeed, the M145F mutant
designed specifically to block the tunnel between zones 2 and 3
lost ∼80% activities for PGE and NGE (SI Appendix, Table S2).
To verify whether zones 1 and 3 are responsible for substrate
access in BmEH catalysis, we developed a mass spectrum method
for capturing the transition state intermediates (SI Appendix, Fig.
Fig. 2. Mass-spectrometric analysis of the covalent intermediates in the
BmEH-catalyzed NGE hydrolysis reaction. (A) Relative activities of mutant
M145F and T241R/L168E compared with the wild-type BmEH. (B–D) Com-
parison of the molecular mass changes of the H267F (B), T241R/L168E/H267F
(C), and M145F/H267F (D) BmEH variants before (black) and after (red) re-
acting with NGE. The theoretical molecular mass increase of the enzymes is
200 Da. Mutations at residue Met-145 severely blocked the formation of the
enzymatic covalent intermediates.
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Table 1. Kinetic parameters for the wild-type, M145A, and F128A BmEH toward the substrate
rac-PGE or -NGE
rac-PGE
rac-NGE
KM, mM
Enzyme
kcat, s−1
KM, mM kcat/KM, s−1·mM−1
kcat, s−1
kcat/KM, s−1·mM−1
Wild type
M145A
F128A
>400*
>9.5*
4.4 0.2
>50†
>50†
N.A.
N.A.
0.37 0.05
0.60 0.06
19.0 2.7
34.1 0 0.4 0.75 0.24
1.3 0.3
1.5 0.5
0.45 0.12
12
5
12
1
45 15
The PGE and NGE concentrations were varied in the ranges of 3–50 and 0.3–10 mM, respectively. N.A., not
available.
*Calculated based on the highest rate detected.
†No saturation was observed. The KM values were beyond the concentration range of substrate. The concentra-
tion of PGE for activity determination was limited by its low solubility.
for the pre-steady-state kinetics analysis could be detected from center. We further used the BmEHF128A–(R)-NPD complex as the
the observed kinetic trace of reactions (SI Appendix, Fig. S7). initial model to perform 80-ns MD simulation calculations to pre-
Nevertheless, the stopped-flow fluorescence experiment showed dict the product-release pathways in the active tunnel of BmEH. As
that the H267F mutation caused relatively slow intermediate
formation and hydrolysis (SI Appendix, Fig. S7). This finding
enabled us to detect the subtle difference between the substrate-
consumption rate and the product-releasing rate of BmEH var-
iants by HPLC in minute scale. As shown in Fig. 3, the F128A/
H267F mutant had a similar pattern of intermediate formation to
that of H267F BmEH, whereas the M145A/H267F mutant be-
haved differently and proceeded at a higher intermediate for-
mation rate. This result suggested that the M145A mutation
could accelerate the formation of enzymatic intermediate and
thereby increase its catalytic activity toward the bulky NGE.
We further solved the crystal structure of BmEHM145A and
BmEHF128A–(R)-NPD complex [NPD, the hydrolyzed product of
(R)-NGE] at resolutions of 1.70 and 2.90 Å, respectively (SI
Appendix, Table S3). Although the overall structures of M145A
and F128A are highly similar to that of the native BmEH (with
rmsd values of 0.186 and 0.239 Å, respectively), the mutations ex-
panded the cavity of zone 2 and made it more accessible for zone 3
(Fig. 4 A–C). In the complex structure of BmEHF128A–(R)-NPD,
the diol moiety of (R)-NPD is located near the boundary between
zones 2 and 3, whereas the naphthyl moiety of (R)-NPD is in the
active cleft of zone 2 but very close to zone 1. However, based on
a structural alignment of other EHs with the BmEH–POA complex,
the epoxide ring should be hydrolyzed around the area between
zones 1 and 2 (mainly by residues Asp-97, Tyr-144, and -203),
whereas the naphthyl ring would sit in the cavity of zone 2 close to
zone 3 and be stabilized by π–π stacking of Trp98. This finding
suggested that there was a 90° conformational rotation of the hy-
drolyzed product (R)-NPD when it was released from the catalytic
shown in Fig. 4D and Movie S1, the product (R)-NPD penetrated
zone 2 to reach zone 3 for exit. This process was accompanied by
dramatic conformational change of residues Trp-98, Leu-132, and
Phe-209. Collectively, these findings indicated a possible catalytic
route for BmEH: The substrate enters through zone 1, then un-
dergoes hydrolysis in zone 2, reaches zone 3, and, finally, exits to
the solvent.
Gram-Scale Synthesis of (S)-Propranolol with M145A. With mutants
F128A and M145A in hand, we turned our attention to NGE
bioresolution. Because F128A was expressed mainly as inclusion
bodies and had lower enantioselectivity (E = 45) than M145A
(E > 100), we chose M145A for organic synthesis. The kinetic
parameters of BmEH WT and M145A toward (R)- and (S)-NGE
were measured (Table 2). The wild-type BmEH and mutant
M145A had similar KM values for the preferred (R)-NGE and
less preferred (S)-NGE substrates. However, in the wild-type
BmEH, the substrate specificity constant (kcat/KM) for the pre-
ferred (R)-NGE was ∼30 times that for the less preferred
(S)-NGE; in mutant M145A, the kcat/KM for the preferred (R)-NGE
was ∼200 times that for the less preferred (S)-NGE. Mutation of
Met-145 to alanine increased both the catalytic activity and the
enantioselectivity for NGE. Indeed, when racemic NGE was
bioresolved at a substrate load of 20 g·L−1, (S)-NGE and (R)-diol
were obtained in 99.4% ee and 91.1% ee, respectively. Compared
with that of wild-type BmEH, the E value of mutant M145A was
markedly improved from 25 to 131. Subsequently, a simple re-
action of (S)-NGE with isopropylamine afforded the desired
(S)-propranolol in 36% yield (1.86 g).
Fig. 3. The enzyme–substrate intermediate formation curve of H267F (A), F128A/H267F (B), and M145A/H267F (C). ●, Concentration of substrate (NGE); ■,
concentration of product (NPD); ▲, concentration of intermediate. Values were calculated by deduction of the remaining NGE and NPD concentrations from
the inputs (25 μM). Reaction conditions were as follows: Each variant at a concentration of 25 μM was mixed with 25 μM (R)-NGE in potassium phosphate
buffer (100 mM, pH 7.0) containing 10% DMSO at 30 °C. Samples were withdrawn at different intervals, mixed with methanol for termination, and analyzed
by RP-HPLC (C18 column). The concentration of NGE and NPD was quantified by the area of corresponding peaks on HPLC.
Kong et al.
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As illustrated in the modified Michaelis–Menten equation, the
catalytic efficiency (kcat) of the enzymatic reaction is determined
by both k2 of the hydrolysis step and k3 of the product-release
step (SI Appendix, Fig. S8). When the product dissociation is pre-
sumed as the rate-limiting step, expanding the product-release
site of active tunnel would increase k3 and thereby enhance kcat
.
The release of product from enzyme is the last step in the cat-
alytic cycle, which is very important for the catalytic perfor-
mance, and the substrate specificity might be partially controlled
by this step (30). Much effort has been made to identify and
engineer the product-release sites of various enzymes. In most
cases, the product release and substrate access share the same
tunnel in proteins (31–33). However, the product-release tunnel
in enzymes such as haloalkane dehalogenases is distinct from the
substrate access tunnel, which sometimes makes the product
release step a rate-limiting bottleneck, thereby providing a new
target for efficient protein engineering (34–36). Previously, there
was no report of an independent product-release site in EHs. In
this work, we have identified a potential product-release site in
BmEH and successfully used this information to engineer
mutants with substrate specificity toward bulky epoxides (Fig. 5).
Considering of the high structural similarity among the super-
family of α/β-hydrolases, this strategy might become an alterna-
tive choice for expanding the substrate scope of enzymes.
Compared with the existing methods for rational protein engi-
neering, targeting the substrate-access or the transition-state sites
(37, 38), the “two birds with one stone” approach described herein
may might be advantageous for increasing the catalytic activity of
the target enzyme and for reducing its product inhibition. With the
rapid growth of new enzyme structures and development of mo-
lecular modeling dynamics software, more and more valuable
enzymes might be modified by this approach.
Fig. 4. The M145A and F128A mutations made zone 2 expand and attach to
zone 3. (A−C) Surface presentation of zones 2 and 3 in wild-type BmEH
(cyan), M145A (yellow), and F128A (gray). (D) Snapshots of MD simulation on
the BmEHF128A–(R)-NPD complex at 0 ns (green), 42 ns (blue), and 80 ns
(yellow). Significant conformation changes took place in residues Trp-98,
Leu-132, and Phe-209 around zones 2 and 3.
Discussion
EHs hydrolyze the racemic epoxides to yield highly enantiopure
epoxides and vicinal diols. However, their application was greatly
hindered by limited substrate scope and low enantioselectivity
and regioselectivity. Previous protein-engineering efforts on EHs
including direct evolution, rational design, and CAST strategy
have generated several mutants with improved enantioselec-
tivities. However, the catalytic efficiency of engineered EHs
on bulky epoxide substrates is still not satisfying. In this work,
we have identified an active tunnel in an EH with unusual
(R)-enantioselectivity and modified it suitably for efficient bio-
resolution of bulky pharmaco NGE. Although the wild-type
BmEH exhibits higher affinity for NGE (KM = 1.3 0.3 mM)
than for less bulky PGE (KM > 50 mM) (Table 1), its efficiency
on NGE hydrolysis was much lower, with an activity decreased by
two orders of magnitude and an E value reduced to ∼25. We
found that this result might be caused by the jammed active
tunnel. The F128A and M145A variants, designed to remove the
steric hindrance in the product-release site, were found to suc-
cessfully expand zone 2 and enable it to more openly commu-
nicate with zone 3. Interestingly, the M145A mutant was also
found to be beneficial for the formation of transition state in-
termediate. The kcat/KM values of F128A and M145A toward
NGE were enhanced by 57 and 32 times that of wild-type BmEH,
respectively. Moreover, we can carry out gram-scale synthesis of
(S)-propranolol with the M145A variant of BmEH. Further
studies to generate an EH library by semisaturation mutations on
the identified hot spots has broadened the substrate scope to
more drug precursors in our laboratory (29).
Materials and Methods
Chemical Reagents. PGE was purchased from TCI. The 2-phenoxyacetamide
was bought from Alfa Aesar. Racemic NGE was prepared by using corre-
sponding phenols and epichlorohydrin as reported (22). (R)- and (S)-NGE
were prepared with a preparative-scale HPLC (Waters) equipped with an
OD-H 10 × 250 mm (Daicel) column. (R)-NPD {(R)-3-[1]-naphthyloxy-propane-
1,2-diol}, the hydrolyzed product of (R)-NGE, was prepared in the laboratory
of J.-H.X. All other chemicals were obtained commercially.
Expression and Purification of BmEH. The wild-type and mutant BmEH from
B. megaterium strain ECU1001 were expressed as N-terminal His-tagged
proteins in Escherichia coli BL21(DE3) as described (22). All mutations were
generated by PCR using the QuikChange method (Strategene) and confirmed
by DNA sequencing. Proteins were purified by using one Ni-NTA columns,
followed by thrombin protease treatment, and one gel filtration column
(Superdex 75 Hiload 16/60; GE Healthcare). Details are described in SI Appendix.
Selenomethionine-substituted (SeMet) BmEH was expressed via the methio-
nine inhibitory pathway (39) and purified similarly to the wild-type protein.
Crystallization, Data Collection, and Structure Determination. Rhombohedral
crystals of BmEH were grown at 20 °C by using the sitting-drop vapor diffusion
method by mixing the protein (20 mg/mL) with an equal volume of reservoir
solution containing 0.5 M LiCl, 0.1 M Tris·HCl (pH 6.5), and 25% (wt/vol) PEG
6000. The SeMet BmEH was crystallized under the same condition. Crystals of
the BmEH–POA complex were prepared by cocrystallization of 5 mM POA in
the protein solution and grown under the condition of 0.2 M Li2SO4, 0.1 M
Table 2. Kinetic parameters of the wild-type and M145A BmEH catalyzed reactions of (R)- and (S)-NGE
(S)-NGE
KM, mM
(R)-NGE
KM, mM
kcat/KM, s−1·mM−1 E [=(kcat/KM)R/(kcat/KM)S]
Enzyme
kcat, s−1
kcat/KM, s−1·mM−1
kcat, s−1
Wild type 0.039 0.005 1.40 0.42
M145A 0.23 0.02 1.62 0.32
0.028 0.009
0.14 0.03
0.75 0.08 0.87 0.32
29.1 3.1 1.01 0.36
0.86 0.33
28.8 10.7
31
206
Reactions were performed in 0.1 M potassium phosphate buffer at pH 7.0 with 10% DMSO as cosolvent and 0.02% Tween 80, and the
(R)- and (S)-NGE concentrations were varied in the range of 0.3–10 mM.
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Fig. 5. A proposed model for bulky (R)-diol production by the M145A and F128A BmEH variants. The M145A or F128A mutation expanded the predicted
product release site of BmEH, which would facilitate the hydrolysis of bulky NGE. Tyrosines (Tyr-144 and -203) and aspartic acid (Asp-97) were served as Lewis
acid and Lewis base, respectively.
Tris·HCl (pH 7.0), and 1.6 M (NH4)2SO4. The M145A mutant was crystallized under
the condition of 0.2 M Li2SO4, 0.1 M Tris·HCl (pH 8.5), 34% (wt/vol) PGE 3000. The
complex of F128A and (R)-NPD was made by cocrystallizing the mutant enzyme
with 5 mM (R)-NGE under the condition of 0.2 M Li2SO4, 0.1 M Tris·HCl (pH 7.5),
and 2.0 M (NH4)2SO4. The hydrolysis of (R)-NGE in the crystallization process
afforded the product (R)-NGE. Before data collection, crystals were soaked in
Paratone-N (Hampton Research) and then flash-cooled in liquid nitrogen.
Diffraction data of the native and SeMet BmEH were collected at the
wavelength of 0.9791 Å by using a MX-225 CCD detector and an ADSC
Quantum 315r detector, respectively, at beamline BL17U of the Shanghai
Synchrotron Radiation Facility. Diffraction data of the BmEH–POA complex,
BmEHM145A, and BmEHF128A–(R)-NPD complex were collected at the wave-
length of 1.5418 Å on a RaxisIV++ imaging plate detector. All datasets were
indexed, integrated, and scaled by using the HKL2000 package (40). The single-
wavelength anomalous diffraction phases were calculated with SHELX C/D/E
(41). A model of BmEH was built automatically by ARP/wARP (42) and manually
adjusted by using COOT (43). Rounds of automated refinement were per-
formed with PHENIX (44). The structure of the BmEH–POA complex was solved
by molecular replacement method using the program PHASER (45). The atomic
models of BmEH, BmEH–POA complex, BmEHM145A, and the BmEHF128A–(R)-
NPD complex have been refined to 1.85, 1.7, 1.95, 1.7, and 2.9 Å, separately.
Crystallographic statistics are summarized in SI Appendix, Table S3. All figures
of the protein models were prepared with Pymol (www.pymol.org/).
and the fluorescence difference of NGE and NPD was observed at 380 nm
through a monochromator. All reactions were performed in 100 mM
potassium phosphate buffer (pH 7.0) containing 10% DMSO at 25 °C with
enzyme concentrations of 25 μM and substrate concentrations of 125 μM.
Volumes of 80 μL were injected from each syringe, and the reported con-
centrations are those in the reaction chamber. A path length of 1 cm was
used throughout the stopped-flow experiments.
Chemoenzymatic Synthesis of (S)-Propranolol. Gram-scale bioresolution of
NGE was conducted by mixing crude enzyme extracted from ∼5 g of fresh cell
pellets and 4 g of racemic NGE in a 500 mL conical flask. The reaction was
terminated at ∼16 h, and the mixture was saturated by adding sodium
chloride followed by extracting with ethyl acetate (150 mL × 3). The re-
sultant mixture with ees of 99% and eep of 91% was purified by using a silica
gel column, affording the enantiomerically pure epoxide (99% ee) and op-
tically enriched diol (88% ee). The obtained enantiopure (S)-NGE was
refluxed in isopropylamine for 48 h, producing 1.86 g of (S)-propranolol (36%
yield after recrystallization).
25
½αꢀD = −8:89ꢀðcꢁ 1:0,ꢁ EtOHÞ, lit:11
½αꢀ2D5 = −8:83ꢀðcꢁ 1:0,ꢁ EtOHÞ:
1H NMR (400 MHz, CDCl3) δ 1.15 (6H, d, J = 6.3 Hz), 2.84–3.01 (3H, m), 3.03
(2H, d, J = 12.4), 4.15–4.23 (3H, m), 6.83 (1H, d, J = 7.4 Hz), 7.40–7.55 (4H, m),
7.84 (1H, m), 8.32 (1H, m).
Activity Assay. The specific activities of BmEH proteins were measured by
monitoring the conversion of substrate by HPLC. Details of the measure-
ments are provided in SI Appendix. Determination of the enantiomeric
excesses of epoxides and diols were performed as reported (22).
Computational Modeling of the BmEH Active Tunnel. Structures of the BmEH–
POA complex and the BmEHF128A–(R)-NPD complex were imported into the
Maestro program (Version 9.2) (46). All MD simulations were performed by
using the Schrodinger 2011-based Desmond package (47). Eighty nano-
seconds for the BmEH–POA complex and 80 ns for the BmEHF128A–(R)-NPD
complex production MD on each system were executed. A total of 100
structures of BmEH extracted throughout the whole MD simulation trajec-
tories were used for CAVER (48) input files. Output from CAVER was finally
visualized and rendered in Pymol. Details of computational modeling are
provided in SI Appendix.
Mass Spectrometric Analyses. The BmEH variants with H267F mutation at
a concentration of 1 mg/mL (0.028 mM) were incubated with substrate NGE
(0.1 mM) for 10 min. The BmEH protein solution was then loaded onto
a homemade capillary column (150 μm inner diameter, 3 cm long) packed
with Poros R2 medium (AB-Sciex). The BmEH proteins were eluted by an
Agilent 1100 binary pump system with the following solvent gradient:
0–100% B in 60 min (A = 0.1 M acetic acid in water; B = 0.1 M acetic acid/40%
acetonitrile/40% isopropanol). The eluted proteins were sprayed into a
QSTAR XL mass spectrometer (AB-Sciex) equipped with a Turbo Electrospray
ion source. The instrument was acquired in MS mode under 5K volts spray
voltage. The protein charge envelop was averaged across the corresponding
protein elution peaks and deconvoluted into noncharged forms by the
BioAnalyst software provided by the manufacturer.
ACKNOWLEDGMENTS. We are grateful for access to beamline BL17U1 at
Shanghai Synchrotron Radiation Facility and thank the beamline staff for
technical support. We also thank Dr. Peter K. Park and Profs. Zhihong Guo
and Ran Hong for helpful discussions. This work was supported by National
Program on Key Basic Research of China Grant 2011CB710800 (to J.-H.X. and
J.Z.), National Grand Project for Medicine Innovation Grant 2012ZX10002006
(to J.Z.), National Natural Science Foundation of China Grant 21276082 (to
J.-H.X.), and a grant from the Open Fund from the State Key Laboratory of
Bioreactor Engineering (to J.Z.).
Stopped-Flow Data Acquisition and Analysis. The stopped-flow experiments
were conducted on an Applied Photophysics model SX20 stopped-flow
spectrofluorimeter fitted with a Xenon lamp. Samples were excited at 310 nm,
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