Evaluating Ylehd, a recombinant epoxide hydrolase from: Yarrowia lipolytica as a potential biocatalyst for the resolution of benzyl glycidyl ether

Article abstract of DOI:10.1039/c8ra00628h

Glycidyl ethers and their vicinal diols are important building blocks in the organic synthesis of anti-cancer and anti-obesity drugs. Ylehd, an epoxide hydrolase from tropical marine yeast Yarrowia lipolytica, was explored for its enantioselective propert

Full text of DOI:10.1039/c8ra00628h

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Evaluating Ylehd, a recombinant epoxide hydrolase
from Yarrowia lipolytica as a potential biocatalyst
for the resolution of benzyl glycidyl ether†
Cite this: RSC Adv., 2018, 8, 12918
a
c
a
a
c
Chandrika Bendigiri, K. Harini,
Sajal Yenkar, Smita Zinjarde, R. Sowdhamini
ab
and Ameeta RaviKumar
*
Glycidyl ethers and their vicinal diols are important building blocks in the organic synthesis of anti-cancer
and anti-obesity drugs. Ylehd, an epoxide hydrolase from tropical marine yeast Yarrowia lipolytica, was
explored for its enantioselective properties by kinetic, thermodynamic and in silico studies. Kinetic
resolution of racemic phenyl glycidyl ether (PGE) yielded (S)-epoxide while for benzyl glycidyl ether
(BGE) (R)-epoxide was obtained, with vicinal diols of the opposite configuration. Amongst the
enantiomers of PGE and BGE, the (S)-selective conversion of benzyl glycidyl ether to its corresponding
diol, (S)-3-benzyloxy-1,2-propanediol while retaining (R)-BGE was most favourable with 95% ee in
2
0 min. Enantioselective conversion of specific enantiomer of BGE to its corresponding diols was
Received 21st January 2018
Accepted 26th March 2018
attributed to the favourable kinetic and thermodynamic parameters as well as to the number and
proximity of water molecules near the base H325 in the active site pocket. The easily available and highly
active Ylehd could be a potential biocatalyst for large scale preparation of pharmaceutically relevant
chiral (R)-benzyl glycidyl ether and (S)-3-benzyloxy-1,2-propanediol.
DOI: 10.1039/c8ra00628h
rsc.li/rsc-advances
through a hydrolytic mechanism as shown in Fig. 1 involving
a catalytic triad comprising of a nucleophile (Asp), a general
Introduction
Chiral epoxides and their corresponding vicinal diols are base (His) and an acid residue (Asp/Glu) with positioning and
essential and valuable synthons for the preparation of enan- activation of the substrate done by two tyrosines. As per the
tiopure biologically active compounds. Chemical routes for the mechanism proposed for murine EH (PDB 1CQZ), catalysis
synthesis of these enantiopure epoxides and diols involve involves a nucleophilic attack by an active site Asp leading to the
asymmetric epoxidation or dihydroxylation of olen substrates formation of an acyl-enzyme intermediate complex. In the next
carried out with catalysts such as chiral salen cobalt complexes step(s), a charge relay system comprising of the acidic residue
1,2
and porphyrin manganese adducts. However, these reactions (Asp/Glu) and base histidine, which abstracts a proton from the
employ toxic chemical reagents and generate by-products that water molecule, thereby activating it and hydrolysing the acyl-
render the process non-ecofriendly. Hence, alternative simple enzyme intermediate resulting in the release of the diol
5
and green biological routes using enantioselective enzymes product.
such as epoxide hydrolase (EH) is a promising approach. EHs
3
In recent times, microbial EH enzymes, especially from fungi
are stable, co-factor-independent and selectively hydrolyse one and yeasts have gained importance in resolution of racemic
enantiomer of racemic epoxide into the corresponding vicinal epoxides. Kinetic resolution of styrene oxide (SO) and its
diol by addition of a molecule of water, leaving the slow reacting derivatives has been well studied using EH from Aspergillus sp.,
enantiomer. Therefore, effective production of optically pure Rhodotorula glutinis and Sphingomonas sp. with high yields and
6
–8
epoxides depends on the appropriate EH as well as the substrate enantiomeric excess (ee) of 98–99.5%. Asymmetric hydrolysis
building block. EHs (EC 3.2.2.3) belonging to the a/b hydrolase of aliphatic epoxides mediated by yeasts have also been
4
superfamily are ubiquitously found in nature. They catalyse the
conversion of epoxides to their corresponding vicinal diols
a
Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune
4
11 007, India. E-mail: ameeta@unipune.ac.in
b
Department of Biotechnology, Savitribai Phule Pune University, Pune 411 007, India
National Centre for Biological Sciences, Bengaluru, India
c
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra00628h
1
Fig. 1 Generalized reaction mechanism for epoxide hydrolase.
12918 | RSC Adv., 2018, 8, 12918–12926
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9
22
reported. Another class of epoxides, the aryl glycidyl(oxiran-2- able to hydrolyse PGE and BGE. In this study, we attempt to
ylmethyl) ethers such as phenyl glycidyl ether (PGE) and understand the basis for enantioselective properties of the
benzyl glycidyl ethers (BGE) and their diols are useful inter- protein by in silico techniques and in vitro assays wherein the
mediates for synthesis of chirally relevant compounds in (S)-selective resolution of BGE to (S)-3-benzyloxy-1,2-
pharmaceutical, agrochemical and ne chemical industries. propanediol has been demonstrated with accumulation of (R)-
Kinetic resolution of PGE and its derivatives has been well BGE. Using steady-state kinetic analysis, thermodynamic
10,11
studied
for the synthesis of b-blockers in cardiovascular studies and molecular dynamic simulations, the probable
therapies. For PGE, while bacterial EHs selectively yield (S)- reason for the enantioselective behaviour of Ylehd has been
epoxide (>99% ee) and (R)-diols (89% ee), the yeast cells of suggested.
Trichosporon loubierii yielded (R)-epoxide with good enantiose-
lectivity (99% ee) and (S)-diols (33% ee) depending on the cell/
substrate ratio. In contrast, benzyl glycidyl ether (BGE) and
Results and discussion
12
its derivatives, important building blocks for anti-cancer and Specic activity of Ylehd with racemic PGE and BGE was found
ꢀ
1
anti-obesity drugs, have been less explored. For example, (R)- to be 18.23 and 25 U mg protein, respectively. No activity
BGE is used as a starting compound in the chemical synthesis of could be detected with styrene oxide (SO) and 2-methyl-3-
1
3
anti-obesity drug, (ꢀ)-tetrahydrolipstatin, marketed as Xenical
phenyloxirane (MPO) as substrates under the given assay
conditions.
14
and of Spiket-P, a potent cytotoxic drug against cancer cells.
The product of the EH reaction with BGE i.e., diol(S)-benzyloxy-
,2-propanediol also has signicant applications. It is used in Steady state kinetics of enantioselective hydrolysis by Ylehd
1
the synthesis of lipophilic aldehydes to inhibit human gastric
lipase (HGL) and pancreatic lipase (HPL) and function as potent
Studies with racemic (rac), (R) and (S) enantiomers of PGE and
BGE with Ylehd suggest a substrate saturation prole as per the
Michaelis Menten kinetics (Fig. S1†). Kinetic constants indicate
15
anti-obesity drugs. Additionally, it is used in the synthesis of
16
the macrolide core of migrastatin, an anti-cancer drug. Thus,
both (R)-BGE and the (S)-diol product have signicant phar-
maceutical relevance. Though resolution of BGE by EH from
microorganisms such as Rhodotorula mucilaginosa and Rhodo-
coccus fascians M022 have been reported, the enantioselectivity
(
rac) BGE as a more suitable substrate for Ylehd. Though the
ꢀ1
turnover (kcat) of PGE (7.34 s ) was higher than that of BGE
ꢀ1
(5.73 s ), the specicity constant (kcat/K
m
ꢀ1 ꢀ1
) was greater for BGE
s ).
ꢀ1 ꢀ1
(38.2 mM
s
) than PGE (12.02 mM
As seen in Table 1, catalytic efficiency (kcat/K
m
) and K
m
values
17,18
is low (3–7 E-value)
and very few reports of EH with high
ꢀ1 ꢀ1
were found to be marginally higher for (R)-PGE (25.5 mM
and 1.32 mM) as compared to (S)-PGE (16.6 mM
s
enantioselectivity towards the pharmaceutically relevant BGE
exist. In recent times attention has been focused on obtaining
high enantioselectivity with high yields, the basis for enantio-
selectivity or the kinetics associated with the resolution of
racemic epoxides has been less explored.
ꢀ1 ꢀ1
s
and 0.93
ꢁ
mM) with a calculated E-value of 1.5 at 30 C in 30 min. This was
slightly lower than the E value of 4.6 for Aspergillus niger EH
which showed slight selectivity in favour of (S)-PGE. Higher E
value of 65 has been observed for TpEH1 from Tsukamurella
paurometabola giving (R)-PGE with enantiomeric excess of more
than 99% ee and 45% yield. BMEH from Bacillus megaterium
ECU1001 preferentially hydrolysed the (R)-PGE yielding (S)-
epoxide and (R)-diol with high enantioselectivity (E ¼ 47.8) and
enantiomeric excess of >99.5% and 55.9% yield.
Amongst the enantiomers, (S)-BGE seems to be the most
suitable ligand for Ylehd both in terms of binding and catalysis.
The K
BGE (0.8 mM). The kcat/K
greater than that for the (R)-enantiomer (4.31 mM
Yarrowia lipolytica NCIM 3589, isolated from oil contami-
nated areas of Mumbai High has been studied for its use in
biodiesel production, biodegradation of brominated alkanes
19–21
and synthesis of nanoparticles.
A protein sequence with
NCBI accession number XP_504164 (named as Ylehd) was
22
cloned, expressed and puried as a 43 kDa protein. Ylehd,
a bifunctional protein, shows epoxide hydrolase activity at pH
8.0 towards aliphatic and aromatic epoxides and promiscuous
m
values for (S)-BGE (0.27 mM) were lower than that of (R)-
haloalkane dehalogenase activity at pH 4.5 towards halogenated
compounds. Preliminary studies suggested that Ylehd was also
ꢀ1 ꢀ1
m
for (S)-BGE (44.7 mM
s ) was
ꢀ1 ꢀ1
s
),
a
Table 1 Kinetic parameters for Ylehd under steady-state conditions
Binding energy from
docking studies (kJ mol
ꢀ1
ꢀ1 ꢀ1
303 K (mM
b
ꢀ1
ꢀ1 ꢀ1
c
Compounds
rac-PGE
)
k
s
)
K
m
(mM)
k
cat (s
)
k
cat/K
m
(mM
s
)
E
ꢀ25.73
ꢀ26.27
ꢀ25.77
ꢀ26.36
ꢀ27.15
ꢀ28.41
17.3 + 0.82
28.11 + 0.73
21.9 + 1.05
21.3 + 0.08
18.2 + 0.47
31.1 + 0.97
0.6 ꢂ 0.009
1.32 ꢂ 0.045
0.93 ꢂ 0.071
0.15 ꢂ 0.002
0.8 ꢂ 0.01
7.34 ꢂ 0.06
12.2 ꢂ 0.78
25.5 ꢂ 0.97
1.5
(
(
R)-PGE
S)-PGE
33.79 ꢂ 0.87
15.66 ꢂ 0.49
5.73 ꢂ 0.54
3.45 ꢂ 0.089
12 ꢂ 0.97
16.6 ꢂ 0.057
38.2 ꢂ 1.08
4.31 ꢂ 0.07
44.7 ꢂ 0.87
rac-BGE
10.37
(
(
R)-BGE
S)-BGE
0.27 ꢂ 0.024
a
b
Kinetic constants calculated by non-linear regression analysis in Microcal Origin. Kinetic studies for all compounds were carried out with 2 mg
c
m
protein except for (R)-BGE wherein 10 mg protein was used. Enantiomeric ratio (E) was calculated as ratio of kcat/K of the fast-acting enantiomer
over the slow acting one.
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resulting in an E value of 10.37, calculated as a ratio of (kcat/K
)
m S
Table 2 Comparative scores of generated models
ꢁ
to (kcat/K ) at 30 C. As seen in Table 1, Ylehd had a preference
m R
Model
number
Energy values
Z-score
from Pros-A
Ramachandran
plot distribution
for (S)-BGE and the E-value for BGE was ꢃ7 fold higher than that
for PGE. It is to be noted that though reports on enantioselective
conversion of PGE exist, little information is available on the
enantioselective conversion of BGE.
ꢀ1
a
(kJ mol
)
3
8
1
1
1
ꢀ55 379.8
ꢀ55 494.4
ꢀ54 134.3
ꢀ55 411.8
ꢀ54 140.3
ꢀ5.51
ꢀ5.02
ꢀ5.35
ꢀ4.78
ꢀ4.88
84, 11.3, 2.7, 2.0
81.3, 13, 3.7, 2.0
84, 11.3, 2.7, 2.0
77.7, 15.7, 4.7, 2.0
79.3, 15.3, 3.3, 2.0
1
2
7
Ylehd hydrolysis of racemic BGE determined by chiral HPLC
In order to validate the enantioselectivity of Ylehd for BGE,
a
Values in the Ramachandran plot distribution are the percentage of
wherein the enzyme hydrolyses the racemic BGE converting the residues in the four quadrants of the plot.
(S) form to the diol while retaining the (R)-enantiomer in the
epoxide form, chiral HPLC analysis of the reaction mix was
carried out. Separation of the two enantiomers was seen with
retention times of 8.3 and 9.3 minutes for (S)-BGE and (R)-BGE,
respectively (Fig. S2†). At the end of the reaction in a short time
period of 20 minutes, the (R)-enantiomer was retained in the
reaction mixture with 95% ee and yield of 10%. The diol formed
in the reaction was seen to retain conguration since (S)-3-
benzyloxy-1,2-propanediol was formed in the reaction while (R)-
BGE was retained as analysed by chiral HPLC. This conrmed
the (S)-selective nature of Ylehd for BGE.
It is to be noted that hydrolysis of the racemic forms would
be majorly inuenced by the differential behaviour of the
individual enantiomers in the active site pocket of Ylehd. Thus,
to study the mode of binding of the enantiomers in the active
site pocket required modelling of Ylehd. As no crystal structure
of the protein is available we attempted to model this bifunc-
tional protein.
ꢀ
1
energy of ꢀ55 379.8 kJ mol and Z score of ꢀ5.51 was selected
for further studies. This model had a typical a/b hydrolase fold
with a cap region exclusively consisting of a helices and a cata-
lytic domain consisting of 7 parallel and one anti-parallel b-sheet
(
Fig. 2a). The topology of Ylehd (Fig. 2b) is similar to that of the
25
general a/b-hydrolase fold EH proteins. The residues forming
the cap region with the conserved tyrosine residues are seen to
consist of only a helices as seen for soluble EH proteins while the
catalytic domain consists of parallel and anti-parallel b sheets
followed by loops bearing the predicted triad residues, namely,
D140, D296 and H325. The nucleophile elbow, formed by G-F-P-
G-S in Ylehd present on a loop between strand b5 and the
following helix a3 contained the nucleophile D140 as is true for
most EH proteins. To further assess the quality of the model,
analysis using Ramachandran plot was also carried out (Fig. 2c).
Number of residues reported to be in the most favoured regions,
in the additionally allowed regions, in generously allowed regions
26
Modelling of Ylehd and docking
Due to high diversity in sequence space and the availability of
a relatively small number of crystal structures of EHs, the
template showing best homology to Ylehd protein was identi-

ed as Murine EH (PDB ID: 1CQZ) with 29% sequence identity
and 92% query coverage. Considering the low sequence simi-
larity of Ylehd with 1CQZ, the second best template with 28%
sequence identity and 40% query coverage from Xanthobacter
autotrophicus exhibiting haloalkane dehalogenase activity (PDB:
1BEE) was additionally considered. Also to be noted that our
earlier studies have shown Ylehd to be a bifunctional enzyme
exhibiting epoxide hydrolase activity with promiscuous hal-
22
oalkane dehalogenase (HLD) activity. Hence, the 2-template
approach was adopted for building the structure of Ylehd to
obtain the model. Both these templates belong to the a/
b hydrolase superfamily and selection of these two templates
Fig. 2 Structure of Ylehd (a) model representing alpha helices (cyan)
and beta sheets (magenta) catalytic triad residues as indicated in the
covers more than 98% of the query sequence to be modelled. In enlarged inset. (b) Protein topology showing shaded region as cap
domain. Residues marked are catalytic triad residues-D140 (a), D296
another study, the structure of EH from Mugil cephalus has been
built with considerable success by homology modelling using
(b), H325 (c), tyrosine residues marked with - Y189 (d), Y258 (e), HGFP
motif A, GFPGS motif : (c) Ramachandran plot distribution A – core
alpha, L – core left-handed alpha, a – allowed alpha, l – allowed left-
handed alpha, -a – generous alpha, -l – generous left-handed alpha, B
two templates, namely, EH from A. niger (1QO7) and A. radio-
23
bacter (PDB: 1EHY). Structure prediction for EH from Agro-
myces mediolanus has also been done using homology – core beta, p – allowed epsilon, b – allowed beta, -p – generous
epsilon, -b – generous beta. Red area corresponds to the ‘core’
regions representing the most favourable combinations of phi (f)–psi
modelling wherein EH from Streptomyces carzinostaticus (PDB:
24
4
I19) was considered as template.
Models (20 numbers) were generated using MODELLER and
ranked based on their energy value, Z-score and Ramachandran
(j) values. Brown corresponds to residues that are comparatively less
favourable with respect to the allowed regions and generously allowed
regions. Yellow corresponds to those residues seen in the ‘disallowed
plot distribution (Table 2). The best model (number 3) with regions’ D140 indicated with arrow.
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ꢀ1
were 84, 11.3 and 2.3%, respectively, while 2% of the residues a difference of 4.8 kJ mol between the two enantiomers at
ꢁ
were in the disallowed regions of the Ramachandran plot. It has 30 C. In contrast, E was signicantly higher for (R)-BGE
a
ꢀ
1
ꢀ1
been shown that a/b hydrolase proteins have a characteristic (75.2 kJ mol ) than that for (S)-BGE (26 kJ mol ) with a differ-
Ramachandran plot wherein the nucleophile of the catalytic triad ence of 49.2 kJ mol suggesting that this difference in the acti-
falls in the disallowed region. This was true even for Ylehd vation energy favours (S)-BGE as the substrate for EH activity of
wherein D140, assumed to be the nucleophile, also falls in the Ylehd at 30 C. The estimated difference in Gibbs free energy of
ꢀ1
26
ꢁ
#
disallowed region. Till date crystal structures of EH from bacte- activation (DG ) for all compounds was <0 suggesting that all the
rial, fungal, plant and mammalian sources reported in the PDB reactions were spontaneous and thermodynamically favourable.
#
database adopt a canonical a/b-hydrolase fold with cap region The DG value was found to be lowest for (S)-BGE
ꢀ
1
and a catalytic domain and similar features have been observed (ꢀ5.69 kJ mol ) as compared to the other enantiomers. From
in the model for Ylehd. Further, to investigate the substrate the temperature dependence of E, values for activation enthalpy
#
#
selectivity of Ylehd, the energy minimized model was used and 9 (DH ) and entropic term (TDS ) were ascertained and were found
aliphatic and 4 aromatic epoxides (Fig. S3†) were docked exibly to be suitable for (S)-BGE (Table 3). Enantioselectivity arises due
and their binding energies compared. The aromatic glycidyl to the difference in the free energy of activation between the
ethers, rac PGE and BGE interacted at the active site with binding enantiomers with contributions from activation enthalpic and
ꢀ1
energy of ꢀ25.73 and ꢀ26.35 kJ mol , respectively. It is to be entropicterms. For BGE, the experimentally determined differ-
#
noted that while kcat values for PGE were much higher than those ential activation free energy (DDG ) values for the individual
ꢀ
1
seen for BGE, Ylehd was found to have a greater affinity towards enantiomers was found to be ꢀ5.43 kJ mol while the calculated
#
ꢀ1
(
S)-BGE as inferred from its Km value. Enantiomers of PGE and DDG values from eqn (2) were found to be ꢀ6.04 kJ mol . Thus,
BGE were found to bind to the same active site with differences the calculated and experimental values were seen to corroborate
seen in the orientation of the ligands. As seen in Table 1, binding with each other. Similar correlations were also studied for PGE
ꢀ
1
#
#
energies of ꢀ26.27 and ꢀ25.77 kJ mol were obtained for (R)- wherein the DDG values from DG values of individual enan-
ꢀ
1
and (S)-PGE, respectively while lower values of ꢀ27.15 and tiomers were found to be ꢀ0.63 kJ mol and calculated value
ꢀ
1
ꢀ1
ꢀ
28.41 kJ mol were obtained for (R)- and (S)-BGE, respectively. from eqn (2) was found to be ꢀ0.79 kJ mol . It can be seen from
# # #
All the four poses show the presence of the catalytic triad residues Fig. 4 that ligands that show greater DDG , DDH and DDS
(
D140, D296, H325), the tyrosines (Y189, Y258) and the key between their specic enantiomers, are more favourable
#
#
residue of the oxyanion hole (F45) as seen in Fig. 3.
substrates for enantioselective resolutions. DDH and DDS
values are positive which indicated that the reaction was spon-
taneous at high temperatures. As suggested by Ottosson et al.
Thermodynamic parameters of Ylehd
(2002), a better transition state binding (low enthalpy) comes
Due to the identical ground state energies of the enantiomers, with a more rigid transition state (low entropy) i.e., an enan-
27
enzyme enantioselectivity can be reduced to the comparison of tiomer favoured by enthalpy is disfavoured by entropy. This
difference in the energetics of transition state of the enantiomers. seems to be the case with (S)-BGE, the enantiomer best acted
Thus, enantioselectivity can be caused by difference in activation upon by Ylehd. Since temperature was seen to play a role on the
free energy between the enantiomers and can be separated into efficiency of the reaction it was noteworthy to nd the racemic
enthalpic and entropic terms of activation. The Arrhenius energy temperature TR wherein both enantiomers are expected to be
of activation (E
a
) was determined for Ylehd with respect to PGE present in equal amounts. The temperature for PGE was fond to
ꢁ
and BGE (Table 3) and found to be marginally higher for (S)-PGE be 300 K (27 C). The E value at this temperature was expected to
ꢀ1
ꢀ1
ꢁ
at 72 kJ mol as compared to (R)-PGE (67.5 kJ mol ) with be 1.0. As per the kinetic studies carried out at 30 C, E value was
fund to be 1.5. Hence it can be clearly seen that the thermody-
namic and kinetic studies seem to correlate well. For BGE, the T
R
ꢁ
was found to be 342 K (69 C) which could be the temperature at
which the E value would be 1.0. Thus, the enantioselectivity
exhibited by Ylehd was temperature dependent which varied
between compounds and could be a parameter to consider while
optimizing the process for resolution of racemic mixtures with
high enantioselectivity and better yields.
Molecular dynamics simulation of Ylehd
The number and proximity of water molecules near the base
H325 at the active site, an important residue in the charge relay,
were studied by molecular dynamic simulations for Ylehd
complexed with (S)-BGE and (R)-BGE. The number of water
molecules (ꢃ11 numbers) are more around (S)-BGE bound to
Ylehd (Fig. 5b) than around (R)-BGE (ꢃ3 numbers) (Fig. 5a).
Furthermore, up to 4 ns the base H325 of Ylehd bound to (S)-
Fig. 3 Ligand protein interaction for enantiomers (a) (R)-PGE, (b) (S)-
PGE, (c) (R)-BGE, (d) (S)-BGE.
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Table 3 Thermodynamic parameters for activation that determine the kinetic resolution of rac-PGE and rac-BGE catalysed by Ylehd
Paper
a
#
#
ꢀ1
#
ꢀ1
#
ꢀ1 ꢀ1
Name of
compound
E
a
DG
DH (kJ mol ),
TDS (kJ mol ),
DS (J mol
K
),
T
R
(K)
ꢀ
1
ꢀ1
(kJ mol
)
(kJ mol ), 303K
303K
303K
303K
(
(
(
(
R)-PGE
S)-PGE
R)-BGE
S)-BGE
67.5 ꢂ 2.3
72.3 ꢂ 3.8
75.2 ꢂ 2.9
26 ꢂ 1.6
ꢀ1.32 ꢂ 0.2
ꢀ0.69 ꢂ 0.04
ꢀ0.23 ꢂ 0.05
ꢀ5.69 ꢂ 0.29
65 ꢂ 3.1
69.8 ꢂ 2.04
72.68 ꢂ 1.98
23.48 ꢂ 2.13
66.32 ꢂ 2.47
70.49 ꢂ 3.09
72.90 ꢂ 2.01
29.17 ꢂ 1.48
219 ꢂ 1.05
233 ꢂ 1.2
240 ꢂ 1.3
96 ꢂ 1.9
300
342
a
All values were calculated as average of 3 sets of experimental replicates and were considered statistically signicant with p < 0.005. Arrhenius
energy (E ) of activation was calculated by plotting the ln k where k is rate constant at each temperature against reciprocal of temperature (1/T)
in kelvin.
a
BGE was closer to the water molecules than that for (R)-BGE been seen in the in vitro assays. However, in case of (R)-BGE,
˚
with a distance as low as 1 A (Fig. 5c). This difference between though d1 and d2 are less than that in (S)-BGE (Fig. S4†), (R)-
the distances may have a role in conversion of (S)-BGE by faster BGE still remains the less preferred substrate. This could be
activation of water molecules by the likely charge relay system of arising either due to high propensity of steric clashes between
D296 and His 325 leading to hydrolysis of acyl-enzyme inter- the interacting atoms or due to the presence of lower number of
mediate complex and release of the diol.
water molecules and greater distance between water and H325
Earlier studies on mutants of Aspergillus niger generated by in the (R)-BGE bound Ylehd. Thus, this study further empha-
directed evolution, have shown that the distance (d) between sizes the role of water and the propensities of water to H325 as
nucleophile and carbon C1 (d1) or carbon C2 (d2) of the bound key contributors to enantioselective property of Ylehd.
epoxide oxirane ring may suggest propensity of attack on the
BGE is an attractive intermediate in synthetic reactions since
ring carbons which in turn could determine the enantiose- the bulkiness of the benzyl group makes it an easy target for
30
lective property or E value of the enzyme. Attack at the C1 would deprotection. Both (R)-BGE and the (S)-diol product have
lead to the (R)-diol product while that at C2 would form the (S)- signicant pharmaceutical relevance. A few studies on resolu-
28,29
diol product.
In this study, docking aer energy minimiza- tion of BGE with variety of microbial strains have been reported
tion showed that in Ylehd bound to (S)-BGE, d1 and d2 were 4.5 in literature (Table 4). It is to be noted that most of these studies
˚
and 5.7 A, respectively while in the case of (R)-BGE bound have been carried out using whole cells. Bioconversions with
˚
docked pose, d1 and d2 are 6.9 and 8.3 A, respectively (Fig. S4†).
Further, the distance trajectories of these bound forms in the
hydrated active site pocket aer molecular docking simulations
of the docked poses were also analysed. In case of (S)-BGE d1 >
d2 up to 4 ns which would facilitate the preference of the (S)-
enantiomer and also formation of the (S)-diol product as has
Fig.
4 Schematic diagram of thermodynamic determinants for
enantioselective property of Ylehd reaction coordinates for Ylehd Fig. 5 Hydrated active site pocket of Ylehd bound with (R)-BGE and
catalysed resolution of PGE (a) and BGE (b) with enantiomers desig- (S)-BGE and distance analysis. Water molecules seen around H325
#
#
#
nated as R (red) and S (black). DG , DH , DS values calculated by with (R)-BGE (a) and (S)-BGE (b) bound to Ylehd after MD simulation
#
#
#
subtracting DG , DH , DS values of the fast acting (R)-PGE enan- (10 ns). Trajectory of distance between H325 of Ylehd (R)-BGE (red)
#
#
tiomer from that of the slower (S)-PGE enantiomer. DDG , DDH , and (S)-BGE (black) bound form and water throughout the simulation
#
#
#
#
DDS values calculated by subtracting DG , DH , DS values of the fast of 10 ns (c). The distance is an average of all the water molecules
acting (S)-BGE enantiomer from that of the slower (R)-BGE present at the active site pocket of Ylehd bound individually to the two
enantiomer.
ligands.
12922 | RSC Adv., 2018, 8, 12918–12926
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Table 4 Strains used for the kinetic resolution of (rac)-BGE
Name of the organism
Type of biocatalyst
Whole cells
E value
13 (R)
%ee
96
Remarks
31
Talaromyces avus
Whole cell
biotransformation aer
growing cells for 72 h and
reaction time of 4 h
Rhodococcus fascians M022
Rhodocccus m-
Whole cells
Whole cells
3–4 (S)
7–8 (R)
45
77
Biotransformation
completion occurs in
12–24 h
Maximum % ee obtained
aer growing cells for 120 h
and 120 h of
ucilaginosa M002 (ref. 17)
32
Aspergillus sydowii
Whole cells
E-value not specied (R)
E-value not specied (S)
E-value not specied (S)
46
biotransformation
32
Trichoderma sp.
Whole cells
60
Maximum % ee obtained
aer growing cells for 120 h
of and growth and aer 25 h
of biotransformation
Resolution of BGE has been
done in this study but
epichlorohydrin remains the
most suitable substrate for
this protein
Agromyces mediolanus
ZJB120203 (ref. 24)
Puried protein
>99
Yarrowia lipolytica NCIM
Puried protein (Ylehd)
10.37 (R)
95
Catalysis brought about by
puried enzyme in 30 min
3
589 (this study)
ꢀ
1
whole cells need incubation times from 25–120 h since biomass agar supplemented with 50 mg ml
generation is the rate limiting step in the process. Also, mass necessary.
transfer of the relatively hydrophobic aryl glycidyl compound
Kanamycin when
and product release in whole cell biocatalysis by intracellular
Spectrophotometric assay for determination of epoxide
hydrolase activity
enzymes or stability of extracellular enzymes, are major factors
contributing to the efficiency of the process. Use of puried
enzymes can eliminate the problems of by-products formed by
side reactions, leading to higher purity and easier downstream
processing.
EH activity was determined spectrophotometrically at 590 nm
32
by measuring the residual epoxide glycidyl ethers. For deter-
mination of kinetic constants varying concentrations of the
substrate were added to the reaction mixture which contained, 2
ꢁ
mg of pure protein in 50 mM Tris pH 8.0, kept at 30 C for 30
Experimental
Chemicals
minutes. One unit of enzyme activity was dened as the amount
of enzyme required for conversion 1 micromole of substrate to
product per minute under the given assay conditions.
Medium components and supplements were obtained from
HiMedia Laboratories Pvt. Ltd (Mumbai, India). Solvents and
compounds were procured from Sisco Research Laboratories Enzyme activity, steady-state kinetics and thermodynamics
Pvt. Ltd (Mumbai, India), Merck Millipore Company (Mumbai,
India), Sigma Aldrich (Missouri, USA) and, racemic, R and S
forms of phenyl and benzyl glycidyl ether were obtained from
TCI chemicals (Tokyo, Japan). All chemicals used in the study
were >99% pure as per the manufacture's specications.
Molecular biology reagents and kits were sourced from Banga-
lore Genei Chemicals (Bangalore, India).
The protein was cloned and expressed in E. coli BL21AI strain.
EH activity was determined spectrophotometrically at 590 nm
33
by measuring the residual epoxide glycidyl ethers.
For determination of kinetic constants, the reaction mixture
containing varying concentrations of the substrate in 50 mM
Tris buffer, pH 8.0, 2 mg of pure protein and were incubated at
ꢁ
3
0 C for 30 minutes. One unit of enzyme activity was dened as
the amount of enzyme required for conversion 1.0 micromole of
substrate to product per minute under the given assay
Strains and media
Yarrowia lipolytica NCIM 3589 cells was grown for 24 h on Yeast conditions.
ꢀ
1
extract Peptone Dextrose (YPD) medium containing (g L ):
Kinetic constants, K
m
, Vmax, turnover numbers (kcat) and the
), were calculated from the initial
yeast extract, 10.0; peptone, 20.0; dextrose, 20.0. Genomic DNA catalytic efficiency (kcat/K
m
extraction was done according to Sambrook and Russell (2001). velocity measurements by Michaelis–Menten equation using
Escherichia coli DH5a and Escherichia coli BL21AI obtained from non-linear curve tting in Origin ver 8.5 (Microcal Ltd, U.S.A).
Invitrogen, (California, USA) were used for clone construction For determination of enantiomeric ratio (E) of the enzyme
and expression studies, respectively. Both strains of E. coli were varying concentrations of pure enantiomers of glycidyl ethers,
routinely grown and maintained on Luria Bertani (LB) broth or were used and calculated as a ratio of the kcat/K
m
of the more
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reactive to the less reactive enantiomer. Energy of activation (E
a
)
SYBYL 7.2 soware. The nal structure was then energy mini-
was calculated for the enantiomers by plotting a graph of ln k mized using the same parameters successively aer construc-
versus 1/T where k is the rate constant at each temperature and T tion of every loop region. Structures were viewed and analysed
#
is the temperature in kelvin (K). Activation enthalpy (DH ), using PyMOL (The PyMOL Molecular Graphics System, Version
#
#
entropy (DS ) Gibb's free energy of activation (DG ) and racemic 1.8 Schr ¨o dinger, LLC).
temperature (T ) were calculated as per the conventional ther-
R
34
modynamic equations as shown below:
Computational enzyme–substrate docking analysis
#
DG ¼ ꢀRT ln k,
(1)
Computational ligand docking studies were carried out using
42
Autodock Version 4.2.6. The homology model of Ylehd protein
obtained in the previous step was used as the receptor and each
of the substrates was used as the ligand. 3D coordinates of the
ligands were downloaded from Pubchem and the ids are as
mentioned in Fig. S3.† Both the protein and ligand were pre-
processed (addition of hydrogens and calculation of charges
for protein and assigning torsions to the ligands) as per the
where R ¼ universal gas constant and k is the rate constant at
03 K,
3
#
DDG ¼ ꢀRT ln E
(2)
For each enantiomer,
#
Autodock protocol. The grid was set for the full protein of size
DH ¼ E
a
ꢀ RT
(3)
ꢀ
1
20 ꢄ 120 ꢄ 120 and a spacing of 0.375 A. Coordinates of
#
#
#
TDS ¼ DH ꢀ DG
(4) Central Grid Point of Maps were 43.915, 43.992 and 43.925.
Docking was carried out with exible ligand for 100 genetic
algorithm runs with a population size of 100 and a maximum of
2
7 000 generations. The interactions in the protein–ligand
43
complexes were analysed using PyMOL and Ligplot.
Resolution of racemic BGE and detection of diol
Racemic benzyl glycidyl ether was added to 50 mM Tris pH 8.0
at a concentration of 20 mM in ethanol and reaction was initi-
ated by addition of 36 mg of puried enzyme, kept at 30 C up to
Molecular dynamic simulation
ꢁ
Molecular dynamics simulations were carried out for the
protein–ligand complexes to observe the presence of water
molecules at the binding site as water is known to play an
important role in catalysis. MD simulations were carried out for
Ylehd protein in complex with (S)-BGE and (R)-BGE (ligands) for
4
0 minutes in shaking water bath. At the end of the incubation
time the reaction mix was extracted 1 : 1 with 95 : 5 hexane:
isopropanol. The organic layer was injected on a Chiralpak IC
column (250 ꢄ 4.6 mm, Daicel, Tokyo, Japan) in a isocratic mode
with the mobile phase 95 : 5 hexane : isopropanol at room
temperature with a ow rate of 0.5 ml min at 240 nm on
Shimadzu UFLC 6-AD. In order to detect the diol product in the
reaction, reaction mixture was lyophilized completely, re-
dissolved in isopropanol and then injected on Chiralpak IG-3
column (250 ꢄ 4.6 mm, Daicel, Tokyo, Japan) in a isocratic
mode with the mobile phase 90 : 05 : 05 n-hex-
10 ns each. The GROMACS molecular modelling soware
ꢀ1
(version 5.5) was employed for performing the MD simulations
of the protein–ligand complexes. Topology les for the protein
and the ligand were created using pdb2gmx. The topology le
for the small molecule ligand was generated using the PRODRG
44
server. A dodecahedron box was utilized for the protein–ligand
complex. The complexes were placed in their respective boxes
such that the distance between the complex and the edge of the
ꢁ
ꢀ1
ane : ethanol : methanol at 15 C with a ow rate of 1 ml min
at 220 nm on Shimadzu UFLC 6-AD.
˚
box was at least 10 A and solvated using the SPC216 water
model. Nineteen sodium ions were added at random positions
into the solvent to neutralize the system. Energy minimization
The PDB database was searched using BLAST-P for suitable to relax internal constraints of the complexes was performed
template to be used in homology modelling. Two templates using the method of steepest descent, with no position
were selected based on identity and query coverage. Multiple restraints applied. Following this, the complexes were subjected
sequence alignment was generated using CLUSTALW. The to 300 ps of equilibration to heat the system to 300 K with
secondary structural features for the query were predicted using position restraints applied. Subsequently, another round of
PSIPRED tool. JOY was used to map the secondary structures equilibration under normal pressure temperature conditions
for the template and annotations were then incorporated in the was performed for 200 ps to bring the atmospheric pressure of
Homology modelling of Ylehd
35
36
37
38
alignment between query and the templates. The alignment the system to 1 bar. A time step of 1 femtosecond was used for
was used to generate the 3-D coordinates of the query using both the equilibration runs. Aer this, 100 ns of production MD
39
MODELLER. The models were validated based on their Ram- under NPT conditions was initiated using the leap-frog inte-
4
0,41
achandran plot distribution and Z-score.
The best model grator with a time step of 2 femtoseconds. V-rescale thermo-
45
46
was energy minimized using the SYBYL 7.2 soware (Tripos Inc. stat and Parrinello–Rahmanbarostat was used to maintain
Ltd), Tripos force eld and Powell's gradient until convergence. temperature and pressure respectively of the system. Coordi-
The energy minimized model was considered for further loop nates were saved every 10 ps. Graphs were generated using the
modelling (wherever templates were unavailable for loops) in Xmgrace plotting tool.
12924 | RSC Adv., 2018, 8, 12918–12926
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1
0 X.-D. Kong, Q. Ma, J. Zhou, B.-B. Zeng and J.-H. Xu, Angew.
Chem., Int. Ed., 2014, 53, 6641–6644.
Conclusions
In this study, we report on the kinetic resolution of glycidyl 11 K. Wu, H. Wang, H. Sun and D. Wei, Appl. Microbiol.
ethers by Ylehd from Yarrowia lipolytica as an epoxide hydrolase
Biotechnol., 2015, 99, 9511–9521.
belonging to the a/b hydrolase family and is the rst report of 12 Y. Xu, J.-H. Xu, J. Pan and Y.-F. Tang, Biotechnol. Lett., 2004,
an enantioselective EH from this yeast. Homology modelling
26, 1217–1221.
using a two-template approach shows that the protein has 13 J. S. Yadav, M. S. Reddy and A. R. Prasad, Tetrahedron Lett.,
a canonical a/b hydrolase fold. The enzyme showed enantiose-
2006, 47, 4995–4998.
lectivity towards (S)-BGE hydrolysing it to its corresponding 14 F. M. Uckun, C. Mao, A. O. Vassilev, H. Huang and S.-T. Jan,
vicinal diol while retaining (R)-BGE up to 95% ee in a short time
Bioorg. Med. Chem. Lett., 2000, 10, 541–545.
period of 20 min. The selectivity of Ylehd for (S)-BGE could be 15 S. Kotsovolou, R. Verger and G. Kokotos, Org. Lett., 2002, 4,
correlated to its kinetic and thermodynamic parameters as well
2625–2628.
as to the number and proximity of water molecules near the 16 C. Gaul and S. J. Danishefsky, Tetrahedron Lett., 2002, 43,
base H325 in the active site pocket. Thus, this study gives an
9039–9042.
insight into the role played by kinetic and thermodynamic 17 M. Kotik, J. Brichac and P. Kysl ´ı k, J. Biotechnol., 2005, 120,
determinants along with the function of water in the enantio-
364–375.
selective conversion of benzyl glycidyl ether catalysed by Ylehd. 18 Q. Ye, J. Bao and J.-J. Zhong, Bioreactor Engineering Research
The results obtained from this study will be used to engineer
and Industrial Applications I: Cell Factories, Springer, 2016.
a robust biocatalyst and optimize the process parameters for 19 G. Katre, C. Joshi, M. Khot, S. Zinjarde and A. RaviKumar,
conversion of glycidyl ethers with higher yields.
AMB Express, 2012, 2, 36.
2
2
2
2
0 A. Vatsal, S. S. Zinjarde and A. R. Kumar, Biodegradation,
2015, 26, 127–138.
Conflicts of interest
1 M. Agnihotri, S. Joshi, A. R. Kumar, S. Zinjarde and
S. Kulkarni, Mater. Lett., 2009, 63, 1231–1234.
2 C. Bendigiri, S. Zinjarde and A. RaviKumar, Sci. Rep., 2017, 7,
There are no conicts to declare.
11887.
Acknowledgements
3 S. H. Choi, H. S. Kim and E. Y. Lee, Biotechnol. Lett., 2009, 31,
1617–1624.
The authors thank BCUD and DST-PURSE programs, SP Pune
University. CB would like to specially thank Department of 24 F. Xue, Z.-Q. Liu, S.-P. Zou, N.-W. Wan, W.-Y. Zhu, Q. Zhu
Science and Technology, Government of India for the nancial
and Y.-G. Zheng, Process Biochem., 2014, 49, 409–417.
support granted under the DST-WOSA scheme (DST-WOS-A LS/ 25 B. van Loo, J. Kingma, M. Arand, M. G. Wubbolts and
64/2013). CB, SY and AR also wish to thank Daicel Chiral
D. B. Janssen, Appl. Environ. Microbiol., 2006, 72, 2905–2917.
Technologies (India) Pvt Ltd for the help rendered in Chiral 26 D. L. Ollis, E. Cheah, M. Cygler, B. Dijkstra, F. Frolow,
2
HPLC analysis. KH was supported by the NCBS bridge post-
doctoral fellowship. KH and RS thank NCBS (TIFR) for infra-
structural support.
S. M. Franken, M. Harel, S. J. Remington, I. Silman,
J. Schrag and others, Protein Eng., 1992, 5, 197–211.
27 J. Ottosson, L. Fransson and K. Hult, Protein Sci., 2002, 11,
1462–1471.
2
8 M. Kotik, A. Archelas, V. Fam ˇe rov ´a , P. Oubrechtov ´a and
V. K ˇr en, J. Biotechnol., 2011, 156, 1–10.
References
1
M. Tokunaga, J. F. Larrow, F. Kakiuchi and E. N. Jacobsen, 29 M. T. Reetz, M. Bocola, L.-W. Wang, J. Sanchis, A. Cronin,
Science, 1997, 277, 936–938.
M. Arand, J. Zou, A. Archelas, A.-L. Bottalla, A. Naworyta
2
S. E. Schaus, B. D. Brandes, J. F. Larrow, M. Tokunaga,
and S. L. Mowbray, J. Am. Chem. Soc., 2009, 131, 7334–7343.
K. B. Hansen, A. E. Gould, M. E. Furrow and 30 N. Kasai, T. Suzuki and Y. Furukawa, J. Mol. Catal. B: Enzym.,
E. N. Jacobsen, J. Am. Chem. Soc., 2002, 124, 1307–1315.
1998, 4, 237–252.
E. J. de Vries and D. B. Janssen, Curr. Opin. Biotechnol., 2003, 31 C. Wei, Y. Chen, H. Shen, S. Wang, L. Chen and Q. Zhu,
4, 414–420. Biotechnol. Lett., 2012, 34, 1499–1503.
S. S. Gill and B. D. Hammock, Biochem. Pharmacol., 1981, 30, 32 M. P. Martins, A. M. Mouad, L. Boschini,
3
4
5
6
7
1
2
111–2120.
B. Schiøtt and T. C. Bruice, J. Am. Chem. Soc., 2002, 124,
4558–14570.
M. H. RegaliSeleghim, L. D. Sette and A. L. Meleiro Porto,
Mar. Biotechnol., 2011, 13, 314–320.
33 R. Kumar, S. I. Wani, N. S. Chauhan, R. Sharma and
D. Sareen, Protein Expression Purif., 2011, 79, 49–59.
34 R. S. Phillips, Trends Biotechnol., 1996, 14, 13–16.
1
C. Morisseau, H. Nellaiah, A. Archelas, R. Furstoss and
J. C. Baratti, Enzyme Microb. Technol., 1997, 20, 446–452.
J. W. Lee, E. J. Lee, S. S. Yoo, S. H. Park, H. S. Kim and 35 T. L. Madden, R. L. Tatusov and J. Zhang, Methods Enzymol.,
E. Y. Lee, Biotechnol. Bioprocess Eng., 2003, 8, 306–308. 1996, 266, 131–141.
S. Wu, A. Li, Y. S. Chin and Z. Li, ACS Catal., 2013, 3, 752–759. 36 J. D. Thompson, D. G. Higgins and T. J. Gibson, Nucleic Acids
8
9
A. L. Botes, C. A. G. M. Weijers, P. J. Botes and M. S. van Dyk,
Res., 1994, 22, 4673–4680.
Tetrahedron: Asymmetry, 1999, 10, 3327–3336.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 12918–12926 | 12925

View Article Online
RSC Advances
Paper
3
3
3
7 D. W. A. Buchan, F. Minneci, T. C. O. Nugent, K. Bryson and 42 G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
D. T. Jones, Nucleic Acids Res., 2013, 41, W349–W357.
8 K. Mizuguchi, C. M. Deane, T. L. Blundell, M. S. Johnson and
J. P. Overington, Bioinformatics, 1998, 14, 617–623.
R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput.
Chem., 2009, 30, 2785–2791.
43 A. C. Wallace, R. A. Laskowski and J. M. Thornton, Protein
Eng., Des. Sel., 1995, 8, 127–134.
9 B. Webb and A. Sali, in Current Protocols in Bioinformatics, ed.
A. Bateman, W. R. Pearson, L. D. Stein, G. D. Stormo and J. R. 44 A. W. Sch u¨ ttelkopf and D. M. F. van Aalten, Acta Crystallogr.,
Yates, John Wiley & Sons, Inc., Hoboken, NJ, USA, 2014, pp.
.6.1–5.6.32.
0 R. A. Laskowski, M. W. MacArthur, D. S. Moss and
J. M. Thornton, J. Appl. Crystallogr., 1993, 26, 283–291.
1 M. Wiederstein and M. J. Sippl, Nucleic Acids Res., 2007, 35,
W407–W410.
Sect. D: Biol. Crystallogr., 2004, 60, 1355–1363.
45 G. Bussi, D. Donadio and M. Parrinello, J. Chem. Phys., 2007,
126, 014101.
5
4
4
46 M. Parrinello, J. Appl. Phys., 1981, 52, 7182.
12926 | RSC Adv., 2018, 8, 12918–12926
This journal is © The Royal Society of Chemistry 2018