Structure-based rational design of a phosphotriesterase.

Area of Science:

• Biochemistry
• Enzyme Engineering
• Environmental Science

Background:

• Pesticide chlorfenvinphos (CVP) poses environmental risks.
• Phosphotriesterases (PTEs) are enzymes capable of hydrolyzing organophosphate pesticides.
• Stereospecificity in enzyme-substrate interactions can limit degradation efficiency.

Purpose of the Study:

• To investigate the structural basis of stereospecificity in Agrobacterium radiobacter phosphotriesterase towards chlorfenvinphos isomers.
• To engineer a variant PTE with enhanced catalytic activity and reduced stereospecificity for CVP hydrolysis.

Main Methods:

• In silico substrate docking of chlorfenvinphos stereoisomers into wild-type Agrobacterium radiobacter PTE.
• Site-directed mutagenesis to create a variant enzyme (W131H/F132A).
• Enzymatic assays to determine the hydrolysis rates of Z-CVP and E-CVP by the wild-type and variant PTEs.

Main Results:

• In silico analysis identified residues F131 and W132 as critical for substrate binding and stereospecificity.
• The engineered variant W131H/F132A exhibited significantly increased hydrolysis rates for both Z-CVP (ca. 480-fold) and E-CVP (ca. 8-fold).
• The variant enzyme demonstrated elimination of stereospecificity, hydrolyzing both isomers efficiently.

Conclusions:

• Residues F131 and W132 in Agrobacterium radiobacter PTE are key determinants of chlorfenvinphos stereospecificity.
• Enzyme engineering can overcome substrate-binding limitations and stereospecificity to enhance pesticide degradation.
• The developed variant PTE offers a promising biocatalyst for the remediation of chlorfenvinphos contamination.