Improving Artificial Enzyme Performance with Distal Mutations

The Challenge: Boosting Artificial Enzymes Beyond Their Limits

Engineered enzymes are a cornerstone of green chemistry, but their catalytic efficiency often falls short of natural enzymes. A key reason for this performance gap is that traditional optimization methods focus almost exclusively on the enzyme’s active site, neglecting the critical role that protein dynamics and distal regions play in catalysis.

To address this, researchers from the Stratingh Institute for Chemistry (University of Groningen) and Zymvol sought to prove that strategically placed mutations far from the active site could unlock new levels of performance.

For this study, the team selected an artificial enzyme based on the lactococcal multidrug resistance regulator (LmrR), which had previously reached, in different studies, a limited level of performance with only active-site mutations. This transcription factor is an ideal scaffold for designing “new-to-nature” enzymes for applications in biocatalysis, bioremediation, and biosensors.

Our Solution

To prove that distal mutations could enhance performance, our researchers used Zymspot, an innovative algorithm within our Zymevolver software. Zymspot’s function is to identify mutations that affect the enzyme’s conformational equilibrium and, in turn, its catalytic performance.

This approach enabled us to quickly identify key areas of the enzyme that influence its behavior. The algorithm filtered millions of possibilities down to a manageable list of just 73 potentially beneficial variants. This computational list was generated in just two days, dramatically accelerating the entire engineering process.

Results & Impact

• Property boost with single mutants
  By screening a small number of mutant enzymes, the team discovered two individual single distant mutations—located more than 11 Å from the active site—that significantly improved the enzyme’s properties. Each of these single mutants showed a +30% increase in activity and a +5-7 °C improvement in thermostability.
  
• Outperformed results with recombinant mutant
  By combining these two beneficial mutations, we created a single recombinant mutant that delivered a 66% higher turnover number and a 14 °C increase in thermostability compared to the parent enzyme.
  
• Validating the power of Zymspot
  The results demonstrate that the use of Zymspot technology provides a distinct strategic advantage in terms of efficiency. A targeted list of promising variants was delivered in just two days, underscoring the significant time and resource savings achievable with our algorithm.

Highlights

• +66% Activity
  A recombinant mutant showed a significant boost in catalytic turnover.
  
• +14 °C Thermostability
  The same mutant demonstrated a substantial increase in thermostability.
• 2 Days for computational list of mutants
  Zymvol’s algorithm quickly identified key mutations, accelerating the engineering process.

✱ This Success Story summarizes the results presented in a peer-reviewed research paper co-authored by scientists from the Stratingh Institute for Chemistry (University of Groningen) and Zymvol Biomodeling.