An S188V mutation alters substrate specificity of non-stereospecific a-haloalkanoic acid dehalogenase E (DehE)

Article abstract of DOI:10.1371/journal.pone.0121687

The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionicacid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by K_m. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.

Full text of DOI:10.1371/journal.pone.0121687

RESEARCH ARTICLE
An S188V Mutation Alters Substrate
Specificity of Non-Stereospecific α-
Haloalkanoic Acid Dehalogenase E (DehE)
1
1
Azzmer Azzar Abdul Hamid , Tengku Haziyamin Tengku Abdul Hamid , Roswanira Abdul
2
3
4
Wahab *, Mohd. Shahir Shamsir Omar , Fahrul Huyop *
1
Department of Biotechnology, Faculty of Science, IIUM, Bandar Indera Mahkota, 25200, Kuantan, Pahang,
Malaysia, 2 Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310, UTM Johor
Bahru, Johor, Malaysia, 3 Department of Biosciences and Health Sciences, Faculty of Biosciences and
Medical Engineering, Universiti Teknologi Malaysia, 81310, UTM Johor Bahru, Johor, Malaysia,
4
Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering,
a11111
Universiti Teknologi Malaysia, 81310, UTM Johor Bahru, Johor, Malaysia
*
roswanira@kimia.fs.utm.my (RAW); fzhutm@gmail.com (FH)
Abstract
OPEN ACCESS
The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halo-
genated compounds but is ineffective against β-halogenated compounds such as 3-
chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed
mutagenesis we show here that introducing the mutation S188V into DehE improves sub-
strate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188
of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-
chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP.
This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue
S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the
backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine
residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate
of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on
Citation: Abdul Hamid AA, Tengku Abdul Hamid TH,
Abdul Wahab R, Omar MSS, Huyop F (2015) An
S188V Mutation Alters Substrate Specificity of Non-
Stereospecific α-Haloalkanoic Acid Dehalogenase E
(
DehE). PLoS ONE 10(3): e0121687. doi:10.1371/
journal.pone.0121687
Academic Editor: Eugene A. Permyakov, Russian
Academy of Sciences, Institute for Biological
Instrumentation, RUSSIAN FEDERATION
Received: October 11, 2014
Accepted: February 3, 2015
Published: March 27, 2015
3
CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by K . This
m
Copyright: © 2015 Abdul Hamid et al. This is an
successful alteration of DehE substrate specificity may promote the application of protein
open access article distributed under the terms of the
Creative Commons Attribution License, which permits engineering strategies to other dehalogenases, thereby generating valuable tools for future
unrestricted use, distribution, and reproduction in any
bioremediation technologies.
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by University
Research Grant Q.J130000.2545.04H01 (FH) and Q.
Introduction
Haloalkanoic acids are used as active components in a wide range of herbicides, insecticides,
J130000.2535.03H01. The funders had no role in
study design, data collection and analysis, decision to plastics, and solvents. The broad application of these recalcitrant pollutants has contributed to
publish, or preparation of the manuscript.
environmental pollution and health problems. Certain enzymes, called dehalogenases, can de-
grade and detoxify haloalkanoic acids and other halogenated compounds and therefore repre-
sent potential solutions for this environmental problem.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0121687 March 27, 2015
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3
CP Binding and Alteration of Substrate Specificity of DehE
Non-stereospecific α-haloalkanoic acid dehalogenase (E.C. 3.8.1.10) catalyses the hydrolytic
dehalogenation of D- and L-2-haloalkanoic acids to yield L- and D-2-hydroxyalkanoic acids, re-
spectively. This enzyme has been isolated and characterised from numerous microbial strains,
including Pseudomonas putida PP3 [1,2], Alcaligenes xylosoxidans spp. denitrificans ABIV [3],
Pseudomonas sp. 113 [4,5], and Rhizobium sp. RC1 [6]. The non-stereospecific α-haloalkanoic
acid dehalogenase E (DehE) was isolated from Rhizobium sp. RC1 by growing this bacterium
in medium containing 2,2-dichloropropionate as the sole source of carbon [7,8]. DehE was ini-
tially characterised along with the stereospecific dehalogenases, DehD and DehL, which were
also isolated from Rhizobium sp. RC1 [6,9]. DehE catalyses the dehalogenation of many α-
haloalkanoic acids but does not catalyse the dehalogenation of β-haloacids [6,10,11]. To date,
only a few strains of bacteria have been shown to produce a dehalogenase that can remove a
chloride ion from the β-carbon of 3-chloropropionic acid (3CP) [12–15]. Jing et al. [13] re-
ported a dehalogenase from Rhodococcus sp. HJ1 that acts on 3-chlorobutyric and
2
,2,3-trichlorobutyric acids, whereas Mesri et al. [14] isolated a haloacid dehalogenase from a
bacterial strain that uses 3CP as its sole source of carbon. In addition, Hamid et al. [16] charac-
terised the ability of β-haloalkanoic acid dehalogenase from Pseudomonas sp. B6P to degrade
3
CP. Recently, computational tools were used to characterise DehE, and it was shown to con-
tain a single active-site cavity within the monomeric enzyme [17]. The DehE active site permits
the binding of 3CP, as well as the α-chlorinated acid compounds D-2CP and L-2CP, at residues
W34, F37, and S188.
Mutagenesis studies by our group have confirmed that the DehE residues W34, F37, and
S188 serve as the substrate-binding residues for D-2CP and L-2CP and that they are necessary
for catalysis [18]. The dehalogenation reaction facilitated by DehE is similar to those catalysed
by other non-stereospecific haloalkanoic acid dehalogenases [5,19]. The enzymatic reaction
utilises a direct attack mechanism that is triggered when D189 activates a water molecule to
generate a hydroxide ion. This ion consequently attacks the C atom of the substrate to liberate
2
the halide ion. Interactions between α-haloalkanoic acid compounds and DehE have recently
been described [17], but there are no published reports concerning interactions between DehE
and β-haloalkanoic acid. In fact, studies concerning non-stereospecific haloalkanoic acid deha-
logenase are rare.
To better characterise the catalytic activity of DehE—specifically its substrate specificity
with respect to β-haloacids—it is important to determine the molecular mechanisms that pre-
vent its catalysis of 3CP. Briefly, enzyme engineering can be conceptually divided into three
categories, namely activity engineering (changing k ), substrate specificity engineering, and
cat
stability engineering (improving resistance to pH, temperature, etc.) [20]. In the current study
we focused on engineering the substrate specificity of DehE by varying spatial interactions,
structural stability, and orientation of the DehE-3CP complex to more closely resemble the
DehE-D-2CP and DehE-L-2CP complexes. This could improve our understanding of the multi-
faceted interaction between DehE and 3CP.
Nakamura et al. [21] investigated the catalytic interactions of L-specific haloalkanoic acid
dehalogenase using a Molecular dynamics (MD) approach. In contrast, Schmidberger et al. [2]
used docking simulation to elucidate binding interactions associated with DehI from Pseudo-
monas putida PP3. Here, our goal was to improve the substrate specificity of DehE towards
3
CP by characterising substrate-docking interactions within DehE-complexes. We used MD
simulations and site-directed mutagenesis analysis. DehE is very specific for 2CP, in this paper
we rationally design the enzyme in order to allow it to react with a non activated chloroacid
namely 3CP. This study may promote the application of protein-engineering strategies to α-
haloalkanoic acid dehalogenases to generate improved reagents for
bioremediation technologies.
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3
CP Binding and Alteration of Substrate Specificity of DehE
Materials and Methods
Data set
The DehE structure used in this study was obtained from the Swiss-Model web server [22] as
described [17]. The structures of D-2CP, L-2CP and 3CP were used as small molecules for the
investigation. These structures were obtained from PubChem, a database maintained by the
National Center for Biotechnology Information [23]. Each complex between DehE and D-2CP,
L-2CP, or 3CP was generated using Autodock Tools version 4.2, as described [17]. Site-specific
mutation of S188 was performed using PyMOL [24], and all complexes including S188V-3CP
mutant were simulated using GROMACS version 4.5.1 [25].
Molecular dynamics simulations
Each of the three complexes involving D-2CP, L-2CP, or 3CP docked to DehE was used as the
starting point for MD simulation. The Gromacs package 4.5.1 adopting the GROMOS 53a6
force field parameter was utilised. Structures were solvated in a cubic box, using periodic bound-
3
ary conditions and the simple point charge water model at a density of 0.998 g/cm . The dimen-
sion of the box was setup with the box edge approximately 10 Å from the molecule (s)
periphery. The water molecules were allowed to relax using the position-restraint procedure.
+
The PRODRG server [26] was used to generate ligand topologies, and 8 Na counter ions were
added to neutralise the total charge of the system. Energy minimisation of enzyme-substrate
complexes was performed using 800 steps of steepest descent. Following this procedure the sys-
tem was equilibrated at constant temperature (303 K) and pressure (1 atm) for 50 ps. Finally,
the equilibrated structure was subjected to production stage whereby NPT ensemble was used
for over 10,000 ps with an integration time step of 2 fs. The non-bonded list was generated using
an atom-based cut-off of 10 Å. Long-range electrostatic interactions were handled using the par-
ticle-mesh Ewald algorithm [27]. All bond lengths involving hydrogen atoms were constrained
using the Lincs algorithm [28]. Trajectory snapshots were collected every 4 ps. Root mean
square deviations (RMSDs), root mean square of fluctuations (RMSFs), total energy, and the dis-
tance between atoms were analysed using Gromacs utilities (g_rms, g_energy and g_dist).
Data analysis
The RMSD plot was used to determine the equilibrated structure, whereas RMSF was used to
identify local flexibility of the system from the MD trajectory. The 10,000 ps RMSD and RMSF
plots were constructed for DehE complexes (DehE-D-2CP, DehE-L-2CP, and DehE-3CP), and
equilibrated structures of the complexes were extracted at 8,000 ps for the substrate-binding
conformation. Total energy plots were generated to determine the energy of DehE-substrate
complexes. At 4,000–4,500 ps, which are the significant range of distance fluctuations for
DehE-3CP complex, the structures were extracted to analyse the atomic movement and
substrate orientation.
Molecular visualisation
Three-dimensional structures of the complexes were visualised using PyMOL [24] and Visual
Molecular Dynamics [29]. Distance variations between substrate-binding residues and sub-
strates were monitored every frame during the course of the simulation.
Site-directed mutagenesis of DehE
Overlapping PCR was used to introduce site-specific mutations. The primers were 5'-ctat-
caccatggccccgcagtc(agc)gatttccaagctc-3' and 5'-gagcttggaaatcgac
PLOS ONE | DOI:10.1371/journal.pone.0121687 March 27, 2015
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3
CP Binding and Alteration of Substrate Specificity of DehE
(gct)tgcggggccatggtgatag-3'. Base changes in the relevant triplets are shown in
bold and the triplets are underlined. The original triplets are given in the parentheses. Each
PCR reaction (50 μl) contained 50 ng pET-22b(+)-dehE, 5 μl 10× reaction buffer, 1.5 μl dNTPs,
1
.25 μl forward primer (125 ng), 1.5 μl reverse primer (125 ng), and 2.5 U Pfu ultra High-
Fidelity DNA polymerase. The thermal cycle program was 95°C for 5 min, followed by 18 cy-
cles of 95°C for 50 s, 55°C for 50 s, and 68°C for 7 min. A final extension step of 68°C for 7 min
was included. The PCR product was treated with DpnI for 1 h and transformed into Escherichia
coli XL1-Blue competent cells. Wild-type and mutant dehE were expressed as described [18].
Bacteria were sonicated, and cell-free extracts containing dehalogenase were harvested for en-
zymatic assays as previously described [6].
Dehalogenase activity assay
The preparation of cell free extracts (CFE) and the measurement of dehalogenase activity were
made as previously described [10]. Dehalogenase activity for the kinetic studies were per-
formed in triplicates by measuring the amount of chloride ions released from 4.7 ml distilled
water containing 0.1 M Tris-acetate (pH 7.6) and 50 μl of 0.1 M 3CP or D-2 chloropropionic
acid (D-2CP) or L-2 chloropropionic acid (L-2CP) as the substrate at 30°C. After 5 min of
equilibration at 30°C, the reaction was initiated by adding soluble cell-free extracts or pure
dehalogenase enzyme to a final volume of 5 ml. Samples (1.0 ml) were removed at 5-min inter-
vals. The released chloride ion in the assay mixture was measured using the colourimetric tech-
nique of Bergmann and Sanik [30] on a UV/VIS spectrophotometer (Perkin-Elmer). Colour
was allowed to develop for 10 min at room temperature and measured at A460nm. Control ex-
periment, lacking enzyme preparation and/or denatured enzyme was included in each set of
assay to detect spontaneous halogen released. Unit of enzyme activity (U) was defined as the
amount of enzyme that catalysed the release of 1 μmol chloride/min. Specific activity (1 μmol
–
–1
–1
Cl Ámin Ámg ) was calculated as the unit of enzyme activity per mg of total protein. Protein
was determined by the biuret procedure with crystalline egg albumin as a standard [31]. En-
zyme specific activity is defined as the μmole of chloride liberated per milligram protein in
1
0 min under the stated conditions.
Analytical methods
High-performance liquid chromatography (HPLC) analysis was performed using Waters 2690
Alliance equipped with separations module with waters 996 Photodiode Array Detector and
Hi-Plex H 300 mm x 7.7 mm column (Agilent Technologies, USA). An Isocratic mobile phase
of 0.005 N H SO with flow rate of 0.6 ml/min was used. Detection was carried out at 210 nm
2
4
at 40°C. Under this condition, the retention time (t ) of 3-hydroxypropionic acid (3HP) and
R
3
CP were 21.85 and 23.92, respectively (Fig. 1).
Protein purification
The purification of dehalogenase protein from cell free extracts of recombinant E.coli strains
was achieved using the method described by Huyop et al. [10].
Results
MD simulation of DehE-d-2CP, DehE-l-2CP, and DehE-3CP
MD simulations were performed for the DehE-D-2CP, DehE-L-2CP, and DehE-3CP com-
plexes. Conformational stability during MD simulations was represented by the RMSD of the
alpha-carbon atoms and three-dimensional structures of DehE-D-2CP, DehE-L-2CP, and
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 1. HPLC profile of 3HP (t
R R
: 21.85) and 3CP (t : 23.92) standards (10 mM each).
doi:10.1371/journal.pone.0121687.g001
DehE-3CP complexes were simulated in explicit water molecules throughout the 10,000 ps of
MD calculations. Water molecules are pertinent for actual simulation of DehE catalysed hydro-
lysis of the 2-haloalkanoic acid substrates since such reaction requires presence of water. Fig. 2
shows the structures reached a stable equilibrium during the simulation. The RMSD for DehE-
D-2CP was steady at ~2.5 Å after 4,000 ps, whereas DehE-L-2CP was steady at ~1.7 Å after
7
1
,000 ps. Overall, the RMSD for both complexes remained fairly constant throughout the
0,000 ps of MD simulation, suggesting that the simulation time was sufficient to obtain equili-
brated forms of the DehE-substrate complexes. The RMSD of the DehE-3CP complex showed
trends that were similar to DehE-D-2CP and DehE-L-2CP. The DehE-3CP complex, however,
recorded a low RMSD value and plateaued at ~2.2 Å after 4,000 ps which implied attainment
of a stable DehE-3CP complex. Therefore, it was appropriate that the equilibrated structures
for all complexes were extracted at 8,000 ps. Confirmation of the substrate binding residues is
established by observing formation of new hydrogen bonds between the enzyme and substrate.
These analyses revealed that the carboxylate group of D- and L-2CP was strongly hydrogen
bonded to residues W34 and the backbone amides of F37, and S188 (Fig. 3). Similar hydrogen
bonding interactions were observed for the DehE-3CP complex which corroborate a previous
report demonstrating that DehE interacts and binds to 3CP through residues W34, F37, and
S188 [17]. It can be inferred that these residues were vital for substrate bonding due to their lo-
cation within the hydrogen bonding distance (< 3 Å) to the substrate moiety.
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 2. Root mean square deviations (RMSDs) of the alpha-carbon positions during 10,000-ps simulations.
doi:10.1371/journal.pone.0121687.g002
The RMSF of each backbone atom within each DehE complex was calculated over the
course of the simulation to assess local flexibility of the system. All DehE complexes showed a
large fluctuation of the N-terminal residue. Residues exhibited dynamic properties within these
complex structures (Fig. 4), with all DehE-3CP and DehE-D-2CP residues fluctuating in a simi-
lar manner. In contrast, residues within the DehE-L-2CP complex fluctuated slightly differently
(
except for the first and last 75 residues). DehE-L-2CP had a more rigid and compact structure,
whereas DehE-D-2CP and DehE-3CP exhibited similar flexibility. Calculating the total energy
of each molecule shed further light on DehE binding preferences. From the first picosecond of
the MD simulation, total energy for each of these complexes was stable (Fig. 5), suggesting high
Ã
5
levels of structural stability. DehE-3CP had a higher total energy (–6.40 10 kJ/mol) than
DehE-D-2CP or DehE-L-2CP, with a difference of ~ 1.00 kJ/mol.
Atomic distance analysis of DehE-d-2CP, DehE-l-2CP, and DehE-3CP
Active-site residues of DehE were previously verified using site-directed mutagenesis, revealing
that W34, F37, and S188 are crucial for substrate binding [18]. Here we measured distances be-
tween these binding residues and substrate in DehE complexes. Calculation of binding dis-
tances throughout simulation time (0 to 10,000 ps) is important to assess the enzyme-substrate
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 3. Molecular visualisation of substrate binding. (A) D-2CP in the binding pocket of DehE. (B)
Hydrogen bonding between D-2CP and W34, F37, and S188 in the DehE-D-2CP complex. (C) L-2CP in
binding pocket of DehE. (D) Hydrogen bonding between L-2CP and W34, F37, and S188 in the DehE-L-2CP
complex. (E) 3CP in the binding pocket of DehE. (F) Hydrogen bonding between 3CP and W34, F37, and
S188 in the DehE-3CP complex.
doi:10.1371/journal.pone.0121687.g003
affinity level of DehE towards 3CP. Nakamura et al. [21] reported that residue K151 of L-DEX
exhibited high affinity towards 2-chloropropionic acid as exemplified by the small atomic dis-
tance (< 3 Å) throughout simulation time. In this study, distances between the carboxylate ox-
ygen (O) of D-2CP or L-2CP and HE1 of W34 (the H atom of the amino acid side chain)
remained fairly constant at ~1.8 Å. Stability of these atomic distances substantiate previous
studies concerning the role W34 plays in binding D-2CP or L-2CP [17]. Similar observations
were made for F37 and S188. Distances between the carboxylate oxygen (O) of D-2CP or L-2CP
and the hydrogen atom of the backbone amide for F37 was ~1.8 Å, as was the distance between
carboxylate oxygen of D-2CP or L-2CP and the hydrogen atom of the backbone amide for S188
(
Fig. 6).
Evaluation of DehE-3CP revealed that the atomic distance between O of 3CP and HE1 of
W34 fluctuated between 1.8 and 4.0 Å. This instability indicates a weak intermolecular
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 4. Root mean square fluctuations (RMSFs) of the alpha-carbon positions of DehE complexes.
doi:10.1371/journal.pone.0121687.g004
interaction, which generally leads to unstable substrate binding and sub-optimal substrate ori-
entation, consequently, the inability to carry out catalysis. Residues, F37 and S188 also dis-
played similar atomic distance fluctuations when bound to 3CP. The atomic distance between
-
O of 3CP and H of F37 varied between 1.8 and 4.0 Å, whereas the distance between O of 3CP
and H of S188 ranged between 1.8 and 3.8 Å (Fig. 6).
For DehE-3CP, significant fluctuations in binding distances were observed between 2,000
and 6,000 ps which were caused by the improper orientation of the 3CP carboxylate within the
-
active site. As seen in Fig. 7, the carboxylate O of the 3CP formed a hydrogen bond with the
hydrogen (H) of the amide group of the S188 backbone from 4,028 ps of the simulation. How-
-
ever, every 8 ps thereafter the O of the carboxylate twisted, relocated, and formed a hydrogen
bond with the hydrogen (H) of the hydroxyl group of the S188 side chain. This repeated hydro-
gen bond relocation was continuously observed during 126 sampled frames. As a result, a sta-
ble DehE-3CP complex is not attained, and catalysis does not occur. These data support to the
argument that DehE does not dehalogenate 3CP because 3CP carboxylate cannot form a strong
hydrogen bond with S188.
Stabilisation of the DehE-3CP complex
At the beginning of the trajectory (t = 0 ps) until ~5,500 ps, RMSD of the S188V-3CP complex
was lower (1.0–2.0 Å) than for DehE-3CP (1.25–2.5 Å) (Fig. 8). Deviation values for both com-
plexes rapidly increased during the initial 5,500 ps, indicating dynamic structural changes dur-
ing the equilibration process. Between 5,500 and 7,500 ps, the RMSDs of S188V-3CP and
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 5. Total energy of DehE complexes during 10,000-ps simulations.
doi:10.1371/journal.pone.0121687.g005
DehE-3CP were stable at ~2.0 Å. After 7,500 ps, however, the deviation value for S188V-3CP
began to decrease, whereas the DehE-3CP RMSD remained at ~2.25 Å. The difference in devia-
tion values between the complexes after 7,500 ps could be attributed to structural changes asso-
ciated with the S188V mutation. Concerning energy plots, S188V-3CP possessed a low total
Ã
5
energy of approximately—8.4 10 kJ/mol (Fig. 9). As such, this mutation significantly reduced
the average total energy of S188–3CP compared with other DehE complexes. This likely re-
sulted from the stabilisation of bonds between the carboxylate group of 3CP and the backbone
of the valine residue within the DehE active site.
Atomic distance analysis revealed that the distance between the carboxylate O of 3CP and
HE1 of W34, H of F37, or H of S188V was maintained at ~2.0 Å until the end of the simulation
(Fig. 10). Binding distances for S188V-3CP did not fluctuate, suggesting that the S188V muta-
tion strengthened the hydrogen bond between DehE and 3CP, thereby generating a more rigid
DehE-3CP complex. The S188V-3CP complex equilibrated more quickly than DehE-3CP, ex-
hibited less structural fluctuations, and was more stable in water.
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 6. Atomic distance analysis of MD trajectories associated with each of the DehE-d-2CP, DehE-l-2CP, and DehE-3CP complexes. (A) Time
course of distances between HE1 of W34 and O of D-2CP (purple line), HE1 of W34 and O of L-2CP (green line), and HE1 of W34 and O of 3CP (red line). (B)
Time course of distances between H of F37 and O of D-2CP (purple line), H of F37 and O of L-2CP (green line), and H of F37 and O of 3CP (red line). (C) Time
-
-
-
-
course of distances between H of S188 and O of D-2CP (purple line), H of S188 and O of L-2CP (green line), and H of S188 and O of 3CP O (red line).
doi:10.1371/journal.pone.0121687.g006
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 7. Interconversion of hydrogen bonds between the 3CP carboxylate and S188 in DehE-3CP complex.
doi:10.1371/journal.pone.0121687.g007
Dehalogenase activity of S188V in cell-free extracts
Dehalogenase activity was assessed by measuring the release of chloride ions from 3CP after ex-
posure to enzyme-containing cell-free extracts. For extracts containing wild-type DehE, no
dehalogenase activity was detected, as expected. For extracts containing the S188V mutant,
however, activity towards 3CP was detected with a specific activity of 2.79 ± 0.01 μmol
–
-1
-1
Cl Ámin Ámg . Controls without adding any cell free extracts prepared from wildtype DehE
and S188V DehE mutant did not show any chloride release in the reaction mixture containing
3
CP as substrate. Cell free extract from S188V DehE mutant was heat denatured at 70°C for 15
min and used in the similar reaction did not show any chloride released from 3CP suggesting
enzyme from S188V DehE mutant can solely act on the 3CP substrate. Summary of enzyme
specific activity for 3CP compared to the activity for D- and L-CP for both wild type and mu-
tant was described in Table 1.
HPLC analysis
The dehalogenation product of 3CP from the enzyme assay reaction using both extracts from
wild type and mutant were evaluated using HPLC. The enzyme assay was allowed to run for 30
min. The enzyme assay reaction using extract from S188V DehE mutant gave rise to 7 peaks
(Fig. 11a). The peak at 21.86 shared similar elution time with 3HP standard, whereas an assay
using extract from wild type DehE did not show a peak at 21.86 suggesting there is no degrada-
tion of 3CP (Fig. 11b). Using enzyme from wild type DehE, the assay was left for more than
one hour and no traces of 3HP was detected in HPLC suggesting there is no enzyme activity oc-
curs in the assay reaction mixture of wild type DehE.
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 8. Root mean square deviations (RMSDs) of the alpha-carbon positions for S188V-3CP and DehE-3CP during 10,000-ps simulations.
doi:10.1371/journal.pone.0121687.g008
Purification of cell free extracts and kinetic analyses
Cell free extracts of wild type DehE and S188V DehE mutant were purified. The fractions had
2
.2 U and 2.5 U enzyme and a specific acivity of 1.9 U/mg and 2.5 U/mg with D,L2-chloropro-
pionic acid (D,L2CP) as substrate, respectively with a recovery of at least 90% in total. Analysis
of the fraction by SDS-PAGE showed that at least 95% of the protein seen was accounted for by
a 32 kDa band (Fig. 12). Current analysis was sufficiently pure for further characterisation.
Characterisation by apparent kinetic analyses of pure extract from mutant and wild type deha-
logenases was performed using 3CP, D-2CP and L-2CP substrates.
Results of the kinetic experiments are shown in Table 2. It was found that S188V mutant
DehE can act on all substrates and does not show any substrate specificity. However, the K re-
m
sults did show significant differences between D-2CP, L-2CP and 3CP with lower K values
m
for L-2CP compared to D-2CP. The calculated kcat values were varied, with D-2CP being the
fastest substrate molecule converted to product, whereas L-2CP was the slowest. The catalytic
efficiencies for the various substrates by DehE mutant showed 3CP has higher specificity con-
stant value than D-2CP.
Discussion
Calculation of the RMSD in a MD simulation serves to assess the stability of the complex struc-
tures as a whole. The absence of asymptotic behaviour in the DehE-3CP complex indicated
that the complex had indeed formed and that structural unfolding did not occur. DehE-3CP
showed similar structural changes as DehE-D-2CP, whereas DehE-L-2CP exhibited only slight
fluctuations and equilibrated much later (after 7,000 ps). Because DehE-L-2CP deviated less
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 9. The total energies of S188V-3CP and DehE-3CP during 10,000-ps simulations.
doi:10.1371/journal.pone.0121687.g009
and had lower RMSD values than complexes involving D-2CP and 3CP, L-2CP likely had stron-
ger interactions with residues W34, F37, and S188, resulting in a more stable complex. These
results explain why DehE catalyses L-2CP more efficiently than D-2CP (Table 2). It has also
been reported that DehE has a smaller K value and a higher specificity constant for L-2CP
m
than for D-2CP [10]. However, comparing structural changes between these two complexes
and DehE-3CP does not sufficiently explain the preference that DehE exhibits towards D- and
L-2CP over 3CP. Analyses of RMSF values and estimations of total energies revealed that the
DehE-3CP complex is more flexible and has higher energy than the other complexes. We attri-
bute these properties to unstable substrate binding due to weak hydrogen bonds and inappro-
priate substrate orientations within the active site. This may explain why DehE cannot degrade
3
CP. Hydrogen bonds formed between polar atoms are generally < 3.0 Å [32] and maintain
correct substrate orientation within the active site as seen in trajectories of MD analyses. Mean-
while, the calculated total energies critically reflect the possible attainment of a catalytically
competent or destabilized conformation of the DehE-3CP complex. With regards to the bind-
ing affinity, it is accepted that combination between low energy of the enzyme-substrate com-
plex and a short binding distance portray high affinity of the enzyme to the substrate.
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 10. Atomic distance analysis of the MD trajectory of DehE-3CP and S188V-3CP complexes. (A)
Time courses of the distance between HE1 of W34 and O of 3CP for DehE-3CP (red line) and HE1 of W34
and O of 3CP for S188V-3CP (blue line). (B) Time courses of the distance between H of F37 and O of 3CP for
DehE-3CP (red line) and of H of F37 and O of 3CP for S188V-3CP (blue line). (C) Time courses of the
-
-
distance between H of S188 and O of 3CP for DehE-3CP (red line) and of H of V188 and O of 3CP for S188V-
CP (blue line)
3
doi:10.1371/journal.pone.0121687.g010
Table 1. Specific activity of enzyme in cell free extract prepared from wild type DehE and S188V mutant DehE.
–
-1
-1
Specific activity (μmol Cl Ámin Ámg )
Substrate
3CP
D-2CP
L-2CP
Wild type DehE
No activity
4.80 ± 0.02
3.79 ± 0.03
5.50 ± 0.04
4.21 ± 0.02
S188V DehE mutant
2.79 ± 0.01
doi:10.1371/journal.pone.0121687.t001
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 11. HPLC profile of 3CP degradation. (a): degradation of 3CP producing 3HP (t
R
21.86). (b) The 3CP
(t
R
: 23.94) was not degraded by the enzyme in cell free extract from the wild type DehE.
doi:10.1371/journal.pone.0121687.g011
Theoretically, a single molecule that contains two electron-rich moieties should position
these moieties as far apart as possible to minimise repulsion and reduce the overall energy of
the molecule. With this principle in mind, the distance between the large β-chloride ion and
the negatively charged carboxylate should be maximised within the 3CP molecule. It is possible
that this distance exceeds the available finite space within the DehE active site, thereby reduc-
ing binding affinity. The binding step is very important during enzyme catalysis as it lowers the
free energy barrier of the substrate, which expedites the forward reaction [32,33]. The 3CP
molecule, therefore, may be too large to form stable hydrogen bonds with DehE. In addition,
Parisini et al. [34] described a complex halogen bonding feature involving σ-hole charge
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 12. SDS-PAGE analysis of cell free extracts and pure proteins. a) Lane 1: Protein markers (Invitrogen); Lane 2: cell free extract of wild type DehE;
Lane 3: S188V DehE mutant. b) Lane 4: pure enzyme extract of wild type DehE (32 kDa) Lane 5: pure enzyme extract of S188V DehE mutant (32 kDa); Lane
6: Protein markers (Invitrogen).
doi:10.1371/journal.pone.0121687.g012
distribution allowing halogen atom to act as an electron donor. Such bonding between small
halogen-substituted ligands and protein receptor can facilitate and expedite
enzymatic dehalogenations.
Polar residues such as serine are associated with local amino acid flexibility and amino acid
disorder [35]. This instability is thought to impart structural specificity [36]. In the case of
DehE-3CP, however, the combination of an expanded 3CP substrate and molecular flexibility
near S188 likely impedes substrate binding. To improve substrate binding in this context, we
sought to stabilise residues surrounding S188 by replacing the S188 side chain with one that
Table 2. Apparent kinetic parameters for hydrolysis of chloropropionate substrates by wild type and mutant enzymes determined under standard
conditions.
-
1
-1
-1
Source of purified enzyme
Substrates
K
cat (s )
K
m
(M)
Specificity constant M sec
Wild type DehE
3CP
Not available
7.76
Not available
Not available
-
-
-
-
-
4
4
4
4
4
4
D-2CP
L-2CP
3CP
(4.8 ± 0.03) x 10
(4.3 ± 0.02) x 10
(4.9 ± 0.02) x 10
(4.5 ± 0.01) x 10
(4.2 ± 0.02) x 10
1.61 x 10
4
12.32
2.86 x 10
4
S188V DehE mutant
12.55
2.56 x 10
4
D-2CP
L-2CP
9.18
2.04 x 10
4
13.11
3.12 x 10
doi:10.1371/journal.pone.0121687.t002
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3
CP Binding and Alteration of Substrate Specificity of DehE
could not participate in hydrogen bonding [37] and that did not interfere with the overall pro-
tein structure. The non-polar valine was chosen to replace S188 because of its comparable mo-
lecular weight, its compatibility with hydrophobic protein interiors [35,38] and is generally
well tolerated [39]. The S188V mutant reduced the intermolecular distance from ~2.5 Å
(
DehE-3CP) to ~2.0 Å (S188V-3CP). A more rigid S188V-3CP structure likely translated into
a well-oriented 3CP molecule within the DehE active site.
According to the hydrophobicity scale of Kyte and Doolittle, substituting the weakly hydro-
phobic serine (relative hydrophobicity = –0.80) with a more hydrophobic valine (4.62) tends to
increase the internal hydrophobicity of the active site, favouring the binding of hydrophobic
substrates [40,41] and promote catalysis [40,42]. Importantly, introduction of the valine residue
eliminated hydrogen bonding between the carboxylate of 3CP and the side-chain moiety. Bonds
were only formed with the hydrogen of the main-chain amide of valine, thereby eliminating the
erratic hydrogen bond interconversion seen with the DehE-3CP complex. Consequently, DehE
substrate specificity and affinity were shifted towards the non-natural substrate 3CP. It was re-
ported that substrate specificity of D-glucose dehydrogenase isoenzyme of Bacillus megaterium
IAM 1030 was successfully altered following substitution with more bulky and hydrophobic
amino acid residues. Of the six mutants created, the G259A variant exhibited the narrowest sub-
strate specificity, whilst retaining comparable catalytic activity and thermostability to the wild-
type enzyme [43]. Most importantly, by increasing the catalytic repertoire of DehE, the enzyme
can degrade a wider range of halogenated acid compounds, potentially providing a ‘one pot’ so-
lution for the successful removal of halogenated compounds from the environment.
Mutation of S188 to valine modified the substrate specificity of DehE and allowed it to deha-
logenate chlorinated propanoic acid in the β-position. An enzymatic assay showed that the mu-
tant DehE degraded 3CP, similar to a proposed pathway by Hughes [44] for
3
-hydroxypropionate (3HP) production by hydrolytic dechlorination of 3CP. This observation
correlated with the higher catalytic efficiencies (specificity constant) value in Table 2 for 3CP.
However, the kinetic values for D- and L-2CP remained somewhat alike for the mutated pro-
tein suggesting possible occurrence of similar enzyme-substrate interaction when subjected to
MD simulation. A possible reaction catalysed by DehE mutant is illustrated in Fig. 13. Degra-
dation of β-substituted halogenated aliphatic acid has also been reported in Pseudomonas sp.
B6P [14]. The 3HP can be obtained from substrate like glycerol [45,46]. However, the current
study implies that 3HP can also be produced from 3CP by dehalogenation process.
Although stability of the enzyme-substrate complex is critical for biocatalysis, other impor-
tant criteria should not be ignored. For example, the presence of water and the orientation of
the catalytic residue are also important for hydrolytic dehalogenation. In this study, we found
that weak substrate bonding associated with 3CP caused instability and prevented degradation.
We confirmed this hypothesis using site-directed mutagenesis. We also calculated distances
and angles between catalytic residues and water molecules for each of the complexes DehE-D-
2
CP, DehE-L-2CP, and DehE-3CP. We found that both the distance and angle between D189
and H of a water molecule remained constant (~1.8Å and 80°) throughout the 10,000-ps, simu-
lation, suggesting that the enzyme is stable (unpublished data) and that degradation is not pre-
vented by the orientation of the catalytic residue relative to water. Similarly, catalytic
orientation has also been discussed regarding the hydrolytic mechanism of L-specific haloalka-
noic acid dehalogenase, L-DEX YL [21].
Conclusions
Advances in computational enzymology have made computational methods more reliable and
yielded more accurate results when studying enzymatic reactions. We demonstrated that DehE
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3
CP Binding and Alteration of Substrate Specificity of DehE
Fig 13. A possible reaction mechanism of hydrolysis catalysed by DehE mutant. Asp189 incorporated with Asn 114 both act as base at the DehE
enzyme structure. This initiate water activation. The nucleophilic attack of the carbon-halogen bond of the substrate takes place and resulted in formation of
intermediate. Finally, the release of halide (chloride ion) from the compound and formation of hydroxylated product can be detected.
doi:10.1371/journal.pone.0121687.g013
does not catalyse the degradation of 3CP because a strong bond is not formed between these
two molecules and thus a stable DehE-3CP complex is not achieved. Weak hydrogen bonding
between DehE and 3CP was evidenced by atomic distance fluctuations between the two mole-
cules. MD simulations and site-directed mutagenesis revealed that substituting S188 with the
small hydrophobic residue valine made the DehE-3CP complex more rigid and elevated affini-
ty for 3CP. The neutral valine side chain neutralised the erratic interconversion of hydrogen
bonding associated with the carboxylate of serine at position 188 and also restricted bonding to
the hydrogen of the main-chain amide in DehE-3CP. Based on our findings, we believe it is
possible to engineer a more versatile DehE through directed mutations within the active site. In
this way, the enzyme-substrate complex can be further stabilised to enhance substrate specifici-
ty and direct affinity towards non-degradable substrates.
Acknowledgments
The authors thank Prof. Rosli Md Illias for protein work, Institute Bioproduct Development
(
IBD)- for HPLC analysis and Mr. Chew Teong Han from Bioinformatic Research Group
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3
CP Binding and Alteration of Substrate Specificity of DehE
(
BIRG)-UTM for technical assistance. AAAH thanks to the Malaysia Government for financial
award under grant number R.J130000.7845.4F611 (FRGS).
Author Contributions
Conceived and designed the experiments: FH AAAH. Performed the experiments: AAAH. An-
alyzed the data: MSS THTAH. Contributed reagents/materials/analysis tools: MSS RAW.
Wrote the paper: RAW.
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