Tài liệu Báo cáo khoa học: Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation pdf

In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the

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

Tel.: +86 10 821 09844

E-mail: wunf@caas.net.cn

(Received 13 September 2010, revised 25

September 2010, accepted 27 September

2010)

doi:10.1111/j.1742-4658.2010.07895.x

Protein thermostability can be increased by some glycine to proline muta-tions in a target protein However, not all glycine to proline mutamuta-tions can improve protein thermostability, and this method is suitable only at care-fully selected mutation sites that can accommodate structural stabilization

In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved

by the experiments The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186–193) was identified as having high conforma-tional fluctuation The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P⁄ G198P) were modeled and analyzed using molecular dynamics simulations The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured The results were consistent with the computa-tional predictions, and the mutant G194P was found to have higher ther-mostability than the wild-type enzyme

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; Tm, the unfolding temperature measured using CD; WT, wild type.

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-tein conformational changes can be studied over small

time increments (in the ps range) [28] By calculating

the root mean square deviation (rmsd) and root mean

square fluctuation (rmsf) values for backbone atoms,

thermally sensitive or conformationally flexible regions

of a protein can be identified [29]

In this study, the structure of Ochr-MPH was

con-structed through homology modeling MDS were

per-formed on the modeled structure to examine the

region with the greatest conformational fluctuation

The only two glycines near this region, G194 and

G198, were selected as mutation targets Structures of

the hypothetical mutants, namely G194P, G198P and

G194P⁄ G198P, were modeled and analyzed using

MDS to test the effect of the mutations; the mutant

G194P was predicted to have increased

thermostabil-ity This prediction was also supported by

experimen-tal data on the thermostability the wild-type (WT) and

mutant proteins expressed in Escherichia coli

Results and Discussion

Three-dimensional model of Ochr-MPH

The three-dimensional (3D) structure of Ochr-MPH

was modeled using the crystal structure of MPH (PDB

ID: 1P9E) obtained from Pseudomonas sp WBC3 as

the template [6] The resulting model for Ochr-MPH is

shown in Fig 1; it can be described as an ab⁄ ba

sand-wich typical of the metallo-hydrolase⁄ oxidoreductase

fold [6] The final model of Ochr-MPH, as determined

using discovery studio 2.5.5 software, possessed good

stereochemical quality with only one residue (Asp112) 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 fluctuation is shown in purple.

software MDS were executed for 5 ns on the three

modeled structures (G194P, G194P⁄ G198P and

G198P) using Gromacs 4.05 [30] The calculated rmsd

and rmsf values are shown in Figs 2 and 3,

respec-tively Similarly to the WT Ochr-MPH, the

conforma-tions of the three mutants became stable during the

MDS after 3000 ps (Fig 2) The average rmsd values

over the final 2 ns were as follows: 0.43 ± 0.01 nm for

WT Ochr-MPH, 0.31 ± 0.02 nm for the G194P

mutant, 0.44 ± 0.03 nm for the G194P⁄ G198P

mutant, and 0.42 ± 0.02 nm for the G198P mutant

During the simulation, rmsf values of residues 186-193

for the mutant G194P were lowest among the WT and

three mutants (Fig 3), indicating that a more stable

conformation was achieved for these residues

Kinetic characterization of WT and mutant

enzymes

The genes encoding the WT and mutant enzymes were

cloned and expressed in E coli After purification, each

of the expressed proteins migrated as a single band, of

33 kDa apparent molecular mass, on SDS⁄ PAGE (Fig 4) Kinetic parameters of WT and mutant enzymes were measured as described in the Materials and meth-ods, and the results are shown in Table 1 All mutants had methyl parathion hydrolase activity, but the mutant G194P had a higher overall catalytic efficiency (kcat⁄ Km) than the other mutants and the WT enzyme The overall catalytic efficiency (kcat⁄ Km) of the mutant G198P was lower than that of the WT enzyme The overall catalytic efficiency (kcat⁄ Km) 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 (T50) was approximately 67C, which is higher than that for the WT enzyme (62C), and for the G194P⁄ G198P (61C) and G198P (54C) mutants, as shown in Table 1, whereas the T50 of G198P was lower than that of the WT enzyme The

T50 of the double point mutant (G194P⁄ G198P) was similar to that of the WT enzyme and between those

of G194P and G198P

To further investigate the thermostability of the WT and mutant enzymes, the unfolding temperature (Tm) was measured using CD spectroscopy The CD spectra

Fig 2 rmsd values during a 5.0-ns MDS for WT MPH and mutant

enzymes (G194P, G194P⁄ G198P and G198P).

Fig 3 rmsf values calculated over the last 2 ns time window for

WT MPH and mutant enzymes (G194P, G194P⁄ G198P and G198P).

Fig 4 SDS ⁄ PAGE analysis of the purified WT MPH and mutant enzymes (G194P, G194P ⁄ G198P and G198P) Lane 1, purified WT MPH; lane 2, purified G194P; lane 3, purified G194P ⁄ G198P; lane

4, purified G198P; and lane 5, protein marker The positions of the molecular mass markers are shown on the right side of the picture.

of the WT and mutant enzymes at 25C were measured

from 190 to 240 nm at pH 7.4 and found to be identical

Then, the enzyme samples were heated from 20 to 86C

and the CD signals of the enzyme were read at 222 nm

using the MOS-450 (Bio-Logic, Grenoble, France) The

Tmvalues, as shown in Table 1, were determined at the

unfolding curves (Fig 6) The mutants of G194P and

G194P⁄ G198P showed Tmvalues that were 3.3C and

0.6C higher, respectively, than that of the WT enzyme

(Table 1) The Tm of mutant G198P was 1.0C 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)1lower than that of the WT enzyme, which indicated that the structure of G194P was more stable than that of the

WT enzyme, as shown in Table 2 The structural stabil-ity induced by the G194P mutant was mainly a result

of the enhanced electrostatic interaction and van der Waals interactions, as the electrostatic and van der Waals energies of the G194P were lower than those of the WT enzyme (Table 2) As the structure of the G194P mutant become more stable than that of the WT enzyme, the rmsf values (calculated by the MDS) of the residues would be reduced, which is demonstrated in Fig 3 As a result, the G194P mutant exhibited better thermostability than the WT enzyme

Table 1 Comparison of properties of the WT (Ochr-MPH) and mutant (G194P, G198P and G194P ⁄ G198P) enzymes K m and k cat values were calculated by nonlinear regression analysis using GRAPHPAD PRISM All values are expressed as mean ± SD, based on three separate experiments.

m (C) T50(C)

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).

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

stored in our laboratory [7] The E coli strains JM109

(Promega, Madison, WI, USA) and BL21 (DE3)

(Nov-agen, Darmstadt, Germany) were used for recombinant

plasmid amplification and protein expression, respectively

The vector pET-30a(+) (Novagen), which introduces a

His6-tag (His-tag; Novagen) at the N-terminus, was

used for gene expression All restriction enzymes were

obtained from TaKaRa (Otsu, Japan) Isopropyl

thio-b-d-galactoside, kanamycin and imidazole were purchased

from Ameresco (Tully, NY, USA) All chemicals were of

analytical grade

Construction of WT Ochr-MPH and mutants

Genomic DNA of Ochrobactrum sp M231 was extracted

using a bacterial DNA extraction kit (Tiangen Biotech,

Beijing, China) according to the manufacturer’s

instruc-tions The gene encoding MPH (GenBank accession no.:

EU596456) was amplified from the genomic DNA using the

PCR; the primers used in this PCR are shown in Table 3

The PCR product of the WT Ochr-MPH sequence was

purified using a gel-extraction kit (Tiangen Biotech),

digested with EcoRI and NotI, then ligated to the

pET-30a(+) vector Site-directed mutagenesis was

per-formed using the overlap-extension PCR method [35] to

generate the corresponding fragments for the following

mutants: G194P, G198P and G194P⁄ G198P The primers

used to construct mutant MPHs using the

overlap-exten-sion method are shown in Table 3 PCR products of the

mutants were also digested with EcoRI and NotI, and then

cloned into pET-30a(+) DNA sequencing was performed

to validate the insert genes at the State Key Laboratory of

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 coliBL21 (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-bind 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⁄ Pi(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

Table 2 The potential energy, van der Waals energy and electrostatic energy of the WT (Ochr-MPH) and mutant (G194P, G198P and G194P ⁄ G198P) enzymes Calculation is based on the force field CHARMm Values are in kcalÆmol)1.

a

The corresponding energy value;bThe energy difference between the protein and the WT MPH.

Table 3 PCR primers for the wild-type (Ochr-MPH) and mutant (G194P, G198P and G194P ⁄ G198P) enzymes.

Enzyme Primer sequence Wild-type MPH a Forward: 5¢-TAGAATTCGCTGCTCCACAA

GTTAGAACT-3¢

Reverse: 5¢-TAGCGGCCGCTTACTTTGGGTTA ACGACGGA-3¢

Mutant MPHb G194P 5¢-CCTGACGATTCTAAACCGTTCTTCAAGGGTGCC-3¢ G198P 5¢-AAAGGTTTCTTCAAGCCGGCCATGGCTTCCCTT-3¢ G194P ⁄

G198P

5¢-CCTGACGATTCTAAACCGTTCTTCAAGCCGG 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.

(40 lgÆmL)1) and 98 lL of 50 mm Tris buffer, pH 8.0 The

reactions were incubated at 37C 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 37C

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

was performed at 37C using nine different concentrations of

methyl parathion, ranging from 1 to 160 lm Each test was

carried out with at least three replicates The Kmand kcat

val-ues were calculated by nonlinear regression using graphpad

prism5.0 (GraphPad Software Inc., La Jolla, CA, USA)

Thermostability assay of WT and mutant

enzymes

All of the purified enzymes were diluted to 120 lgÆmL)1

with 50 mm Tris buffer (pH 8.0) The diluted enzymes were

incubated at various temperatures, from 45 to 75C, for

10 min Immediately after heating, the enzymes were placed

on ice for 30 min The residual MPH activity was measured

using the assay described above, and at least three samples

were run in parallel

CD spectrometry

CD measurements of the WT and mutant enzymes were

performed using a MOS-450 CD spectrometer (Bio-Logic,

France) equipped with a TCU-250 Peltier-type

temperature-control system Spectra were recorded from 190 to 240 nm

using a 1-mm cell and a bandwidth of 1 nm The unfolding

curves were measured at 222 nm, from 20 to 86C, using

the temperature scan mode with a gradient of 1CÆmin)1

The measurements were performed in 10 mm NaCl⁄ Pi(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 Softsoft-ware 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

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

300 K, with the heavy atoms and Ca atoms fixed Finally,

a 5-ns MDS was performed on the whole system at 300 K All bond lengths were constrained using the LINCS algo-rithm [38] The cut-off value for van der Waals interac-tions was set at 1.0 nm, and electrostatic interacinterac-tions were calculated using a particle mesh Ewald algorithm [39] The time step of the simulation was set at 2 fs, and the coordi-nates were saved for analysis every 1 ps Post-processing and analysis were performed using standard Gromacs tools and customized Perl scripts

Structure energy calculations

The structures of the WT and mutant enzymes were minimized using the discovery studio 2.5.5software with the ‘Minimization protocol’ The minimization algorithms of the Steepest Descent and Conjugate Gradient methods were used with a Generalized Born implicit solvent model [40] The run steps of each mini-mization were set at 5000 steps Then, the potential energy, van der Waals energy and electrostatic energy for the structures of the WT and mutant enzymes were determined with the discovery studio 2.5.5 software using the calculate energy protocol

Acknowledgements

This work was supported by grants from National High Technology Research and Development Program

of China (863 Program, 2007AA100605)

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