Enhanced thermostability of methyl parathion hydrolase
from Ochrobactrum sp. M231 by rational engineering of a
glycine to proline mutation
Jian Tian, Ping Wang, Shan Gao, Xiaoyu Chu, Ningfeng Wu and Yunliu Fan
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China
Introduction
Methyl parathion is an organophosphate pesticide that
has been used extensively in agriculture [1–7]. It is an
acetylcholinesterase inhibitor – a neurotoxin that can
cause wide-scale environmental pollution [1,4,8,9].
Methyl parathion hydrolase (MPH; EC 3.1.8.1), iso-
lated from the soil bacterium Ochrobactrum sp. M231
(Ochr-MPH), is a 33-kDa organophosphate hydrolase.
Although it degrades methyl parathion efficiently, it
has poor thermostability, which can affect the applica-
tion of the enzyme [7]. Having previously cloned the
mph gene from Ochrobactrum sp. M231 [7], we sought
to increase the thermostability of this MPH using pro-
tein engineering.
The two main protein-engineering strategies that can
be used to increase protein thermostability are rational
design and random mutagenesis [10–12]. Of these two
Keywords
methyl parathion hydrolase; molecular
dynamics; proline theory; thermostability
Correspondence
Ningfeng Wu, Biotechnology Research
Institute, Chinese Academy of Agricultural
Sciences, 12 Zhongguancun South Street,
Beijing 100081, China
Fax: +86 10 821 09844

Abbreviations
3D, three dimensional; MDS, molecular dynamics simulations; MPH, methyl parathion hydrolase; Ochr-MPH, methyl parathion hydrolase
from Ochrobactrum sp. M231; rmsd, root mean square deviation; rmsf, root mean square fluctuation; T
50
, the temperature at which the
enzyme lost 50% of its activity; T
m
, the unfolding temperature measured using CD; WT, wild type.
FEBS Journal 277 (2010) 4901–4908 ª 2010 The Authors Journal compilation ª 2010 FEBS 4901
methods, computer-assisted rational design is an inex-
pensive and straightforward route to engineer
improved protein thermostability, because site-directed
mutagenesis techniques have been well developed
[10,11,13,14]. However, many factors affect protein
thermostability, and no clear-cut guarantees of success
exist [11,12,15–17].
Glycine, the only amino acid that lacks a b-carbon,
has the highest conformational entropy [18], while pro-
line can adopt only a few configurations and has the
lowest conformational entropy [19,20]. A glycine to
proline mutation could therefore decrease the confor-
mational entropy of a protein and lead to stabilization
[21–25]. This ‘proline theory’ was proposed by Suzuki
et al. [22,23] and has been used successfully to improve
the thermostability of many enzymes [25–27]. How-
ever, not all glycine to proline mutations can improve
protein thermostability, and this method is suitable
only at carefully selected mutation sites that can
provide structural stabilization [13,19,25,27].
Using molecular dynamics simulations (MDS), pro-

located out of the generously allowed regions in the
Ramachandran plot.
MDS to predict the effect of mutations on protein
stability
MDS were performed on the modeled structure of
Ochr-MPH using gromacs
4.05 [30]. The rmsd values
of the backbone atoms for Ochr-MPH are shown in
Fig. 2, for which the reference structure was the struc-
ture obtained from the equilibration step performed
immediately before the MDS run. The conformation
of Ochr-MPH became stable during the MDS after
3000 ps (Fig. 2).
rmsf values reflect fluctuation at individual residues
– a higher rmsf value indicates less stability [31–33]. As
shown in Fig. 3, residues 186-193 of Ochr-MPH gave
the highest rmsf values. These residues are located at
the protein surface, in a loop region, between a
b-strand and a-helix (shown in Fig. 1). Two glycine
residues, G194 and G198, lie just beyond the C-terminal
end of this region. G194 and G198 were therefore cho-
sen as target sites for the glycine to proline mutation.
The 3D models of the three mutants (G194P,
G194P ⁄ G198P and G198P) were constructed using the
standard mutation protocol of the discovery studio
Fig. 1. Ribbon plot of the three-dimensional structure of Ochr-
MPH, using Pseud-MPH (PDB ID: 1P9E) as the template. Key resi-
dues in the active site are shown in green, and the two Zn ions are
shown in silver. Gly194 is shown in red, and Gly198 is shown in
yellow. The region (residues 186–193) with greatest conformational

⁄ K
m
)
than the other mutants and the WT enzyme. The overall
catalytic efficiency (k
cat
⁄ K
m
) of the mutant G198P was
lower than that of the WT enzyme. The overall catalytic
efficiency (k
cat
⁄ K
m
) of the double point mutant
(G194P ⁄ G198P) was similar to that of the WT enzyme
and between those of G194P and G198P.
Thermostability of WT and mutant enzymes
The thermostability of the WT and mutant enzymes
was determined by measuring residual activity after
incubation for 10 min at various temperatures (Fig. 5).
The temperature at which the G194P mutant lost 50%
of its activity (T
50
) was approximately 67 °C, which is
higher than that for the WT enzyme (62 °C), and for
the G194P⁄ G198P (61 °C) and G198P (54 °C)
mutants, as shown in Table 1, whereas the T
50
of

unfolding curves (Fig. 6). The mutants of G194P and
G194P ⁄ G198P showed T
m
values that were 3.3 °C and
0.6 °C higher, respectively, than that of the WT enzyme
(Table 1). The T
m
of mutant G198P was 1.0 °C lower
than that of the WT enzyme.
These experimental results indicate that replacing
G194 with proline enhances the thermal stability of
Ochr-MPH; however, replacing G198 of Ochr-MPH
with proline did not improve the thermostability.
The thermostability of the double point mutant
(G194P ⁄ G198P) was similar to that of the WT enzyme
and between those of G194P and G198P. These experi-
mental results are in agreement with the MDS results.
The results suggest that determining regions of higher
conformational fluctuation using MDS is a powerful
method to guide selective mutation of glycine to pro-
line to decrease conformational fluctuation, thereby
increasing thermostability.
Structure energy of WT and mutant enzymes
The structure energies of WT and mutant enzymes were
also calculated with the CHARMm force field [34] using
the software discovery studio
2.5.5. The potential
energy of the G194P mutant was 33.7 kcalÆmol
)1
lower

m
(lM
)1
Æmin
)1
) T
m
(°C) T
50
(°C)
Ochr-MPH 252.8 ± 12.64 76.25 ± 4.10 3.32 ± 0.34 67.0 62
G194P 454.70 ± 20.89 64.48 ± 3.41 7.05 ± 0.70 70.3 67
G198P 153.70 ± 6.30 92.70 ± 4.55 1.66 ± 0.15 66.0 54
G194P ⁄ G198P 288.80 ± 10.96 82.73 ± 4.16 3.49 ± 0.31 67.6 61
Fig. 5. Thermostability of WT and mutant (G194P, G194P ⁄ G198P
and G198P) enzymes. The thermal stability of the enzymes was
determined by monitoring residual enzymatic activity after incuba-
tion for 10 min at various temperatures. Enzymatic activity was
then assayed using the standard enzyme assay. Data points corre-
spond to the mean values of three independent experiments.
Fig. 6. Temperature-induced unfolding measured using CD spec-
troscopy for WT MPH and mutant enzymes (G194P, G194P ⁄ G198P
and G198P).
Enhanced thermostability of methyl parathion hydrolase J. Tian et al.
4904 FEBS Journal 277 (2010) 4901–4908 ª 2010 The Authors Journal compilation ª 2010 FEBS
Materials and methods
Bacterial strains, plasmids, restriction enzymes
and chemicals
The bacterium Ochrobactrum sp. M231 was isolated from
the soil at a pesticide factory in Tianjin, China, and

Crop Genetic Improvement, Chinese Academy of Agricul-
tural Sciences (Beijing, China). The correct plasmids for the
WT and mutant enzymes were then transformed into
E. coli BL21 (DE3) for expression [36].
Purification and quantification of recombinant
WT Ochr-MPH and mutants
The N-terminus of each resulting recombinant protein was
fused to a His6-tag that enabled purification using a Ni-ni-
trilotriacetic acid His-bindÔ resin column (Novagen),
according to the manufacturer’s instructions. As the
obtained protein exhibited high concentrations of imidaz-
ole, the protein was desalted with 50 mm Tris buffer (pH
8.0) to determine the kinetic parameters and with 10 mm
NaCl ⁄ P
i
(pH 7.4) to determine the protein thermostability.
The purified proteins were stored at )20 °C in aliquots
until use. The purity of the proteins was analyzed by
SDS ⁄ PAGE followed by staining with Coomassie Brilliant
Blue (R250; Amersham Pharmacia Biotech, St Albans,
UK) [36]. The concentrations of the purified proteins were
determined using the Bio-Rad Protein Assay Kit (Bio-Rad,
Hercules, CA, USA).
Standard enzyme assay
MPH activity was determined by measuring the release of
the product, p-nitrophenol, from the substrate, methyl
parathion [5,8]. The assay mixture (150 lL) contained 2 lL
of 2 mgÆmL
)1
methyl parathion, 50 lL of purified protein

G194P 5¢-CCTGACGATTCTAAACCGTTCTTCAAGGGTGCC-3¢
G198P 5¢-AAAGGTTTCTTCAAG
CCGGCCATGGCTTCCCTT-3¢
G194P ⁄
G198P
5¢-CCTGACGATTCTAAA
CCGTTCTTCAAGCCGG
CCATGGCTTCCCTT-3¢
a
The restriction sites EcoRI and NotI, introduced in the forward
and reverse primers, respectively, are underlined.
b
The oligonu-
cleotide sequence for the forward primer only is shown, and muta-
tion sites are indicated by underlined sequences.
J. Tian et al. Enhanced thermostability of methyl parathion hydrolase
FEBS Journal 277 (2010) 4901–4908 ª 2010 The Authors Journal compilation ª 2010 FEBS 4905
(40 lgÆmL
)1
) and 98 lLof50mm Tris buffer, pH 8.0. The
reactions were incubated at 37 °C for 6 min. The absor-
bance of the liberated p-nitrophenol was measured at
405 nm. One unit of activity was defined as the amount of
enzyme required to liberate 1 lmol of p-nitrophenol per
minute at 37 °C.
Determination of kinetic parameters
Purified enzymes were diluted with 50 mm Tris buffer, pH
8.0, to a final concentration of 12 lgÆmL
)1
. The MPH assay

i
(pH
7.4) using a protein concentration of 3 lm.
Homology modeling of Ochr-MPH
The tertiary structures of Ochr-MPH and the mutants
(G194P, G198P and G194P ⁄ G198P) were modeled using
MODELER, a component of the discovery studio soft-
ware suite v2.5.5 (Accelrys Software Inc., San Diego, CA,
USA). The X-ray crystallographic structure of MPH (PDB
ID: 1P9E) obtained from Pseudomonas sp. WBC3 Pseud-
MPH [6] was used as the template, as it had the highest
sequence identity (98%) with the candidate sequence (Ochr-
MPH). To ensure that the modeled structure was realistic,
the values for the w and u angles of their Ramachandran
plots were checked using the discovery studio software
suite.
MDS
MDS were performed using Gromacs v4.0.5 [30], imple-
menting the Gromos 96.1 (53A6) force field [37]. The ini-
tial structure was solvated with a simple point-charge
model of water in a box with a volume of
90 · 90 · 90 A
˚
3
. A sufficient number of Cl
)
ions were
added to neutralize the positive charges in the system. The
system was then subjected to a steepest descent energy
minimization, and the 30-ps MDS was performed at

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