Identifying and engineering a critical amino acid residue to enhance the catalytic efficiency of Pseudomonas sp. methyl parathion hydrolase
Methyl parathion hydrolase (MPH) that hydrolyzes a wide range of organophosphorus pesticides can be used to remediate land polluted by the pesticides. Here, the catalytic efficiency of methyl parathion hydrolase from Pseudomonas sp. (WBC-3) was enhanced by searching and engineering a critical site far away from the binding pocket. In the first round, a four-site mutant with a modest increased catalytic efficiency (3.2-fold kcat/Km value of the wild type) was obtained with random mutagenesis. By splitting and re-combining the four substitutions in the mutant, the critical site S277, was identified to show the most significant effects of improving binding affinity and catalytic efficiency. With further site-saturation mutagenesis focused on the residue S277, another two substitutions were discovered to have even more significant decrease in Km (40.2 and 47.6 μM) and increased in kcat/Km values (9.5- and 10.3-fold of the wild type) compared to the original four-site mutant (3.0- and 3.2-fold). In the three-dimensional structure, residue S277 is located at a hinge region of a loop, which could act as a “lid” at the substrate entering to the binding pocket. This suggests that substitutions of residue S277 could affect substrate binding via conformational change in substrate entrance region. This work provides a valuable protocol combining random mutagenesis, site-saturation mutagenesis, structural and bioinformatics analyses to obtain mutants with high catalytic efficiency from a screening library of a modest size (3200 strains).
KeywordsMethyl parathion hydrolase Directed evolution Site-saturation mutagenesis Catalytic efficiency Structural and bioinformatics analyses
This work was supported by the 1000 Plan of China (K2069999) and NSFC of Jiangsu Province (BK20151126), NSFC (51603089) to FX, National Natural Science Foundation of China (21406089) to HQY.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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