The mutation at H254 of organophosphorus hydrolase increases the substrate specificity of profenofos

Originally the Organophosphorus hydrolase (OPH, EC 3.1.8.1), isolated from Falvobacterium ATCC27551, was an enzyme published to hydrolyze the easter bond between phosphate and nitro-phenol of parathion, however the molecular structure relationship between the leaving group of the substrate and the binding domain of enzyme still needs to be elucidated. By using error-prone PCR, a mutation library was generated and over 2000 colonies were screened by adding 0.2 mM profenofos to the expression library. A mutant, named 0602B, had 2.1-times degradation activity higher than the wild-type by the whole cell assay. 0602B DNA and wild-type DNA were constructed to the Escherchia.coli. expression vector, enzyme from both strains were purified, and kinetic parameters were analyzed, respectively. Our results showed that the Kcat of mutant 0602B for profenofos was 35.7 + 3.2(n=3) sec^-1 which was average 15.8-fold higher than that of OPH wild type, but the Kcat of mutant 0602B for parathion was 165.25 sec^-1 5-fold lower than that of wild type. Furthermore the mutant 0602B was subjected to do DNA sequence and was analyzed with the wild type DNA and protein sequences. The result showed that 0602B has only one mutation site at the amino acid residue Histidine 254. Compared the protein structure of 0602B with that of OPH wild type (PDB:1EYM) by Discovery Studio 2.5 computer program, His254 is located at the activity site in the hydrophobic core. Our result suggests that the mutation may change the structure of leaving group binding region of substrates and the affinity of profenofos and parathion. Our study will apply to hydrolyze the large leaving group of oranophosphate such as chloropyrophos.