Scoring beyond the Active Site: Distal Mutations

Enzymes are the change-makers of the biological world, orchestrating countless chemical reactions that drive life as we know it. And at the heart of every enzyme lies a crucial spot where the magic happens: the active site.

This is where the enzyme and its substrate come together, catalyzing transformations that are essential for life.

For years, enzyme engineers have focused almost exclusively on this active site, aiming to optimize its function and enhance catalytic activity. And rightly so – after all, it's the hotspot where the action happens.

But what if we told you that there's more to enzyme engineering than just the active site?

Consider the big picture: distal mutations

For some time now, research has shed light on the importance of regions further away from the active site – known as distal regions.

These distant areas play a critical role in shaping the overall conformation and activity of the enzyme, and can have a profound impact on the enzyme's overall performance, influencing its catalytic activity, stability, and specificity.

Enzymes are dynamic molecules, constantly in motion and interacting with their surroundings. The final conformation and activity of the enzyme depend on a network of intramolecular interactions, many of which extend beyond the active site. By targeting these distal regions, enzyme engineers can fine-tune the enzyme's dynamics and unlock its full potential.

Think of it like a game of football: the coach not only focuses on its star striker, but also ensures the optimal performance of the entire team to win a match.

In this same way, enzyme engineers are beginning to recognize the importance of considering the bigger picture, instead of focusing solely on the active site, overlooking the broader impact of distal regions on enzyme function.

Computation-guided engineering of distal mutations in an artificial enzyme

In a recent study, ZYMVOL team members have participated together with researchers from the University of Groningen to demonstrate the potential of targeting distant regions of artificial enzymes to improve their catalytic performance, offering insights into how protein dynamics can be leveraged to enhance biocatalysis efficiency.

Moreover, our team strived to test out the potential that computational tools have in this area, paving the way for more efficient and sustainable biocatalysis.

By screening just a small number of mutant enzymes resulting from Zymevolver -the enzyme engineering software developed at Zymvol- and Zymspot -its utility to depict distal hotspots-, we discovered two single mutations that significantly increased both the enzyme's activity and its stability at high temperatures.

From an initial selection of 73 predicted variants, two variants with mutations distant more than 11 Å from the active site showed increased catalytic activity in a new-to-nature reaction. Recombination of these variants resulted in a 66% higher turnover number and 14 ℃ higher thermostability.

So, the next time you think about enzyme engineering, remember to look beyond the active site. It may be where the magic happens, but it's the distal regions that hold the key to unleashing the full potential of enzymes.

That’s how we can obtain the best possible performing enzymes; and a similar sense of joy as if scoring a goal!