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Supercharged Phosphotriesterase for improved Paraoxon activity.
Jacob Kronenberg1, Dustin Britton1, Leif Halvorsen2
1Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, Brooklyn, New York 11201, USA.
Protein Engineering, Design & Selection : PEDS
|September 18, 2024
Summary
Supercharging improved phosphotriesterases (PTEs) for neutralizing organophosphates (OPs). A negatively supercharged PTE variant showed enhanced catalytic activity, while a positively supercharged variant did not, informing future enzyme engineering.
Area of Science:
- Biochemistry
- Protein Engineering
- Enzyme Catalysis
Background:
- Phosphotriesterases (PTEs) neutralize toxic organophosphates (OPs).
- PTEs exhibit limited thermostability and solubility, hindering their application.
- Protein supercharging enhances protein solubility and thermostability via surface charge mutations.
Purpose of the Study:
- To engineer improved PTE variants using computational supercharging.
- To assess the impact of supercharging on PTEs' thermodynamic stability and catalytic activity against OPs.
Main Methods:
- Employed a computational protein supercharging algorithm (Rosetta).
- Generated and characterized negatively and positively supercharged PTE variants.
- Assessed thermodynamic stability and catalytic efficiency across a temperature range (25°C-55°C).
Main Results:
- A negatively supercharged PTE variant (+12 net charge) exhibited increased catalytic activity from 25°C to 55°C with comparable thermostability.
- A positively supercharged PTE variant (-14 net charge) showed reduced thermostability and catalytic efficiency.
- Supercharging effects on PTEs varied significantly with charge direction.
Conclusions:
- Negative supercharging is a viable strategy for enhancing PTE catalytic efficiency.
- This study provides insights into designing thermostable and highly active PTEs for organophosphate detoxification.
- Findings guide future enzyme engineering efforts for improved biocatalysts.
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