An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, OPHC2.
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.
Chu
Xiao-yu
XY
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, China.
Tian
Jian
J
Wu
Ning-feng
NF
Fan
Yun-liu
YL
D019943:Amino Acid Substitution; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D004220:Disulfides; D004795:Enzyme Stability; D006358:Hot Temperature; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D044205:Pseudomonas pseudoalcaligenes / Q000201:enzymology*
D004220:Disulfides; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases; D003545:Cysteine