Improving Catalytic Activity and Thermal Stability of Methyl-Parathion Hydrolase for Degrading the Pesticide of Methyl-Parathion

Abstract

Pesticides are indispensable in today’s agriculture. Methyl-parathion hydrolase (MPH, E.C.3.1.8.1) could hydrolyze organophosphorus pesticides, including methyl-parathion. MPH could rehabilitate soil and water resources contaminated by organophosphorus pesticides. However, natural MPHs generally exhibited a low tolerance to high temperatures and low catalytic efficiency. In this study, we improved the catalytic efficiency toward methyl-parathion and the thermal stability of the MPH from Pseudomonas sp. WBC-3 through saturation mutagenesis and fusion with self-assembling amphipathic peptides (SAP). The experimental characterization showed that compared to the wild-type enzyme, the kcat/Km of the mutant T271S yielded by saturation mutagenesis was increased by 224.3% compared to the wild-type MPH. T50 and Tm of SAP3-MPH with an SAP fused at the N-terminus were increased by 6.2°C and 6.0°C, respectively. Compared to the wild-type enzyme, T271S fused with SAP3 at the N-terminus (SAP3-T271S) exhibited a 2.1-fold increase in kcat/Km without significantly affecting the thermal stability. The simultaneous improvement of the catalytic efficiency and thermal stability of MPH would be beneficial for its application in the degradation and detection of organophosphorus pesticides. Furthermore, our study provides a potential combination strategy for the design of the other enzyme preparations of pollutant degradation.

Coupling Peptide-Based Encapsulation of Enzymes with Bacteria for Paraoxon Bioremediation

“…The MPH T 50 was found to be at ∼27.5 °C as it had lost more than 50% of its activity above this temperature (Figure c). In comparison, a recent work showed higher thermal stability of Zn-reconstituted MPH . This suggests that while the Mn-reconstituted enzyme exhibited increased activity toward paraoxon in comparison to the Zn-reconstituted MPH, particularly in the range of bacterial growth relevant to its conjugation with bacteria, it presents lower stability at higher temperatures.…”

“…In comparison, a recent work showed higher thermal stability of Zn-reconstituted MPH. 54 This suggests that while the Mn-reconstituted enzyme exhibited increased activity toward paraoxon in comparison to the Zn-reconstituted MPH, particularly in the range of bacterial growth relevant to its conjugation with bacteria, it presents lower stability at higher temperatures. Moreover, after incubation at 28 °C, MPH lost 50% of its activity.…”

Elucidating the Enzymatic Mechanism of Dihydrocoumarin Degradation: Insight into the Functional Evolution of Methyl-Parathion Hydrolase from QM/MM and MM MD Simulations

“…Methyl-parathion hydrolase (MPH) is a xenobiotic-degrading enzyme capable of cleaving the P–O bond in methyl-parathion (Figure b), leading to the formation of less toxic byproducts. − As depicted in Figure , the active site of MPH consists of two zinc ions, bridged by Asp255 and a hydroxide ion. The zinc ion further from the solvent is labeled as Zn α , which is coordinated by residues Asp151, His152, and His302.…”

Enhanced salt-tolerance of Bacillus subtilis glutaminase by fusing self-assembling amphipathic peptides at its N-terminus

“…In our previous study, for the first time, the thermal stability of Pseudomonas aeruginosa lipoxygenase was improved by fusing SAPs to the enzyme N-terminus ( Lu et al, 2013 ). To date, the fusion with SAPs has been successfully used to improve the thermal stabilities of different enzymes, such as polygalacturonate lyase ( Zhao et al, 2018 ), L-asparaginase ( Zhao et al, 2019 ), nitrile hydratase ( Liu et al, 2014 ), and methyl parathion hydrolase ( Shi et al, 2022 ). Compared to the wild-type enzyme, the enzymes fused with SAPs exhibited bigger particle sizes in solutions, suggesting that the oligomerization may account for the enzyme stabilization through the enhanced intermolecular hydrophobic interactions ( Lu et al, 2013 ).…”

“…In our previous study, for the first time, the thermal stability of Pseudomonas aeruginosa lipoxygenase was improved by fusing SAPs to the enzyme N-terminus (Lu et al, 2013). To date, the fusion with SAPs has been successfully used to improve the thermal stabilities of different enzymes, such as polygalacturonate lyase (Zhao et al, 2018), L-asparaginase (Zhao et al, 2019), nitrile hydratase (Liu et al, 2014), and methyl parathion hydrolase (Shi et al, 2022).…”