Using Mutability Landscapes To Guide Enzyme Thermostabilization

Area of Science:

• Protein engineering
• Biocatalysis
• Enzyme technology

Background:

• Thermostabilizing enzymes while maintaining activity and enantioselectivity is vital for biocatalysis.
• Rational design, computational methods, and directed evolution are common strategies.
• 4-oxalocrotonate tautomerase (4-OT) is a potential biocatalyst for pharmaceutical synthesis but can be thermolabile.

Purpose of the Study:

• To explore an alternative mutability-landscape approach for enzyme thermostabilization.
• To identify mutations that increase the melting temperature (T_m) and half-life of 4-OT.
• To assess the impact of these mutations on enzyme activity and enantioselectivity.

Main Methods:

• Utilized a mutability-landscape approach to screen for beneficial mutations in 4-OT.
• Identified single mutations (R11Y, R11I, A33D) that enhance thermal stability.
• Introduced these mutations into an existing enantioselective 4-OT variant (M45Y/F50A).

Main Results:

• Identified three single mutations increasing 4-OT melting temperature (T_m) by up to 20°C and half-life at 80°C by up to 111-fold.
• Triple-mutant variants exhibited up to a 39°C increase in T_m.
• No loss of catalytic activity or enantioselectivity was observed in the improved variants.

Conclusions:

• The mutability-landscape approach is effective for thermostabilizing enzymes.
• This method successfully enhanced the thermal stability of 4-OT without compromising its catalytic properties.
• The findings support the use of mutability-landscape-guided protein engineering for developing robust biocatalysts.