Document Detail


Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230.
MedLine Citation:
PMID:  16734778     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.
Authors:
Irene Horne; Xinghui Qiu; David L Ollis; Robyn J Russell; John G Oakeshott
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  FEMS microbiology letters     Volume:  259     ISSN:  0378-1097     ISO Abbreviation:  FEMS Microbiol. Lett.     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-05-31     Completed Date:  2006-08-25     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  7705721     Medline TA:  FEMS Microbiol Lett     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  187-94     Citation Subset:  IM    
Affiliation:
CSIRO Entomology, Canberra ACT, Australia.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acid Substitution
Base Sequence
Catalytic Domain / genetics
DNA, Bacterial / genetics
Isoflurophate
Methyl Parathion
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Parathion
Phosphoric Monoester Hydrolases / chemistry*,  genetics,  metabolism*
Recombinant Fusion Proteins / chemistry,  genetics,  metabolism
Rhizobium / enzymology*,  genetics
Sequence Homology, Amino Acid
Substrate Specificity
Chemical
Reg. No./Substance:
0/DNA, Bacterial; 0/Recombinant Fusion Proteins; 298-00-0/Methyl Parathion; 55-91-4/Isoflurophate; 56-38-2/Parathion; EC 3.1.3.-/Phosphoric Monoester Hydrolases; EC 3.1.3.-/phosphorylphosphatase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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