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).
ID: ndkZhou54
Submitter: Shu-Ching Ou
Submission Date: March 25, 2019, 3:02 p.m.
Version: 1
Number of data points | 160 |
Proteins | Methyl parathion hydrolase |
Unique complexes | 20 |
Assays/Quantities/Protocols | Experimental Assay: Specific Activity ; Experimental Assay: kcat/Km ; Experimental Assay: kcat ; Experimental Assay: Km ; Derived Quantity: SD of kcat/Km ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km ; Derived Quantity: SD of Specific Activity |
Libraries | Variants for MPH_MP |
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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