Applied Microbiology and Biotechnology

, Volume 88, Issue 1, pp 125–131

An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, OPHC2

Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-010-2738-5

Cite this article as:
Chu, X., Tian, J., Wu, N. et al. Appl Microbiol Biotechnol (2010) 88: 125. doi:10.1007/s00253-010-2738-5

Abstract

OPHC2, a methyl parathion hydrolase (MPH) from Pseudomonas pseudoalcaligenes C2-1 (CGMCC 1150), can degrade a wide range of organophosphate pesticides. Compared with other MPHs, OPHC2 exhibits high thermostability. Its thermostability mechanism, however, remains unknown. In the present study, sequence analysis demonstrated that two cysteines (Cys110 and Cys146) exist in OPHC2, but not in other MPHs. The three-dimensional structural model of OPHC2 performed by computer-assisted homology modelling revealed a potential stacking network with residues Cys110 and Cys146, which probably formed an intramolecular disulfide bond. Furthermore, both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and thiol-titration analyses indicated that OPHC2 contains a disulfide bond. Substitution of the disulfide bond-forming cysteines with alanine, leucine or methionine residues substantially decreased the thermostability of OPHC2, suggesting that disulfide bond formation affects conformational stability. These results, combined with three-dimensional structural modelling, demonstrated that the formation of a C110-C146 disulfide bond may stabilise the conformation of OPHC2, contributing to its thermostability.

Keywords

Methyl parathion hydrolase Disulfide bond Site-directed mutagenesis Thermostability 

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Xiao-yu Chu
    • 1
  • Jian Tian
    • 1
  • Ning-feng Wu
    • 1
  • Yun-liu Fan
    • 1
  1. 1.Biotechnology Research InstituteChinese Academy of Agricultural SciencesBeijingChina

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