Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230.
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.
Horne
Irene
I
CSIRO Entomology, Canberra ACT, Australia.
Qiu
Xinghui
X
Ollis
David L
DL
Russell
Robyn J
RJ
Oakeshott
John G
JG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D007531:Isoflurophate; D008743:Methyl Parathion; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010278:Parathion; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D007531:Isoflurophate; D008743:Methyl Parathion; D010278:Parathion; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases