Two strategies to engineer flexible loops for improved enzyme thermostability

Area of Science:

• Enzyme engineering
• Protein stability
• Biocatalysis

Background:

• Flexible sites in enzymes are targets for stability engineering.
• The rigidifying flexible sites (RFS) strategy has limited success due to poor candidate selection.
• Understanding mutation effects in flexible loops is crucial for enzyme design.

Purpose of the Study:

• To identify effective mutation candidates within flexible loops of Escherichia coli transketolase (TK) for enhanced stability.
• To compare the efficacy of a "back to consensus mutations" approach with computational design using Rosetta.
• To develop strategies for improving enzyme thermostability and performance.

Main Methods:

• Applied two parallel strategies: "back to consensus mutations" and Rosetta computational design (ΔΔG calculations).
• Generated and experimentally characterized 49 single variants of Escherichia coli transketolase.
• Assessed thermostability, half-life, specific activity, and catalytic efficiency (kcat) of variants.

Main Results:

• Identified three single variants (I189H, A282P, D143K) with increased thermostability compared to wild-type TK.
• A combined variant (A282P with H192P) showed a 3-fold improved half-life at 60°C and 5-fold increased specific activity at 65°C.
• The Rosetta program achieved 65.3% qualitative prediction accuracy for stability changes.

Conclusions:

• Both investigated strategies are effective for guiding mutation candidates to flexible loops in enzymes.
• The identified mutations and strategies hold potential for engineering stability in other enzymes.
• This work contributes to a better understanding of enzyme rigidification for improved biocatalytic applications.