Directed evolution to re-adapt a co-evolved network within an enzyme.

Area of Science:

• Biochemistry
• Enzyme Engineering
• Protein Science

Background:

• Targeted active-site saturation mutagenesis identified transketolase single mutants with improved activity towards glycolaldehyde (GA) or propionaldehyde (PA).
• Attempts to combine these single mutants into double mutants resulted in unexpected losses of specific activity and protein stability.

Purpose of the Study:

• To identify beneficial mutations within co-evolved residue networks to enhance transketolase activity towards propionaldehyde (PA).
• To overcome the destabilizing effects observed when recombining previously identified single mutants.

Main Methods:

• Statistical Coupling Analysis (SCA) identified a network of nine co-evolved residues crucial for transketolase function.
• A mutant library was created by combining D469 variants with natural mutations at seven co-evolved residues.
• Synergistic effects of mutations at D469, E498, and R520 on PA activity were investigated.

Main Results:

• A triple mutant cluster at D469, E498, and R520 showed synergistic activity towards PA.
• The D469S and R520Q combination synergistically improved k(cat) 20-fold for PA, creating the most active transketolase mutant to date.
• While some mutations affected expression and solubility, strategic recombination with natural variants enhanced overall enzyme performance.

Conclusions:

• Recombining active-site mutants can destabilize critical co-evolved networks.
• Beneficial single mutants can be retained and improved by recombining them with natural variants within these networks.
• This approach yields highly active and potentially more stable transketolase variants for industrial applications.