Tài liệu Báo cáo khoa học: Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum

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

Tel.: +86 10 821 09844

E-mail: [email protected]

(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 mutations 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; T50, the temperature at which the

enzyme lost 50% of its activity; Tm, 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