Multiscale Simulations Reveal Distal Allosteric Reprogramming of Pocket Dynamics and Dual Hydrolytic Pathways in...

Area of Science:

• Biochemistry
• Enzyme Engineering
• Computational Biology

Background:

• Methyl parathion hydrolase (MPH) is crucial for detoxifying organophosphorus compounds.
• Balancing catalytic activity and thermostability in MPH is a significant challenge.
• Engineered variants MPHase-m5a and MPHase-m5b show improved performance via distal mutations, but mechanisms are unclear.

Purpose of the Study:

• To elucidate the mechanisms by which distal mutations enhance both catalytic activity and thermostability in MPH.
• To understand how remote mutations influence enzyme dynamics and the active site microenvironment.
• To guide the rational design of novel detoxifying enzymes.

Main Methods:

• Multiscale simulations were employed to investigate the molecular dynamics of MPH variants.
• Analysis focused on allosteric networks, active-site pocket dynamics, and substrate binding.
• Enzyme flexibility and temperature-dependent stability were assessed.

Main Results:

• Distal mutations remodel long-range allosteric networks, reorganizing active-site dynamics.
• Allosteric remodeling enhances substrate binding to catalytically competent sites.
• A dual hydrolytic mechanism involving Znα-bound water or bridging hydroxide was identified.
• Long-range allosteric communication modulates enzyme flexibility and thermostability.
• MPHase-m5a achieves thermostability via local rigidification; MPHase-m5b preserves dynamics for efficiency.

Conclusions:

• Distal mutations in MPH act through long-range allosteric networks, not local effects.
• Allosteric remodeling is key to simultaneously enhancing catalytic activity and thermostability.
• Distal allostery represents a viable design principle for engineering detoxifying hydrolases.