Easier, better, faster enzyme discovery

Welcome to Plenty of Room!

Plenty of Room is your guide to the cutting-edge news related to molecular machines.

Today we jump into the deep waters of enzyme engineering, so brace yourselves!

New here? Just go ahead and subscribe! Already subscribed? Help a friend or a colleague save some time and share this with them!

Let’s get into it now.

Easier, better, faster enzyme discovery

Enzymes pretty much enable the entirety of life. Natural evolution shaped these powerful biocatalysts to carry out an incredible range of reactions, and of course scientists wanted to get in on the action. Enzyme engineering typically uses directed evolution, where enzymes undergo rounds of random mutagenesis, followed by screening for improved variants. It’s a powerful method, but it has a downside: it’s slow and often inefficient because most random mutations are unproductive or outright harmful. This means large libraries of variants and tons of screening. So, how do we make this whole process better?

The authors of today’s paper had an idea. They proposed a targeted approach that enriches productive mutational paths, in practice guiding the process toward beneficial mutations while avoiding destabilizing ones. For their test, the researchers used Kemp eliminase, an enzyme that catalyzes a reaction with no natural counterpart (a bit crazy). It’s a well-established model for studying proton transfer in enzymatic reactions, and the authors wanted to show how their new method could rapidly optimize its activity. And spoiler alert: it worked.

Let’s get into the meat of the paper: how they did it. The authors focused on productive mutations, those that improve enzyme function without compromising stability. They removed destabilizing mutations from the library generation process, effectively “pruning” the mutational space to focus on paths that lead to more functional enzymes. This allows the evolutionary process to focus on exploring beneficial mutations without being bogged down by less promising variants. The team achieved this by using computational design tools and gene synthesis techniques:

1. Modeling Protein Structure and Stability: They used the Rosetta Protein Modeling Suite to predict which mutations would harm or help the enzyme. Almost half of the possible mutations were filtered out because they were predicted to be negative!

2.  Library construction: With the remaining productive mutations, they built DNA libraries, introduced them into E. coli, and then tested the activity of the mutated enzymes.

Using this approach, they ran five rounds of evolution on Kemp eliminase, each time refining the enzyme with targeted mutations. In this way, they managed to boost the activity of the enzyme by over 400-fold! This is a crazy improvement when compared to the 17 rounds of evolution required for similar improvements in previous studies.

But the study didn’t stop there. The researchers also explored how different evolutionary paths can lead to similar results. They recombined their newly optimized enzyme with another version of Kemp eliminase evolved in a separate pathway. This allowed them to examine how different combinations of mutations affect enzyme performance. This analysis revealed that different mutations can still lead to highly optimized enzymes, showing just how flexible and complex the enzyme’s fitness landscape is.

This was a very cool study, especially for someone like me that is quite new to enzyme engineering! Their novel approach enhances speed and efficiency of evolution, so that you can obtain similar results with less rounds of evolution (team less experiments here). This approach reduces time and cost for enzyme evolution, and this is great news for different fields:

• Drug Discovery: of course, the potential of a faster, cheaper road to therapeutics is always huge, and worth thinking about. It could be interesting to see if something like this can be applied to the pathway for the production of small molecule drugs, or even cell-free production of biologics.

• Industrial Biocatalysis: engineered enzymes hold great promises for green chemistry, where engineered enzymes are used to catalyze specific reactions under environmentally friendly conditions.

But honestly there is a lot more in this paper, so I’d definitely recommend giving it a read!

In other news:

• A better look at GBPs: Guanylate-binding proteins (GBPs), are involved in defending against intracellular pathogens. In this study, the researchers discovered the structure of human GBP1 and its coat formation on bacterial membranes, by using cryo-EM. This study provides a clearer understanding of GBP1’s role in intracellular immunity. Cool paper with cool figures!

• Finding good problems: How can you find good scientific problems to solve? Not just ones that make you publish in prestigious journals, but ones which are inherently important or impactful? That is the topic of this essay. Definitely worth a read and thinking about it! It’s an interesting read, especially since it’s from 2009 and it doesn’t seem like things have improved since!

• Beautiful, unique viruses: This very cool study analyzed 67,715 predicted viral protein structures from 4,463 eukaryotic viruses and found that 62% are structurally unique with no known homologues. Of the remaining 38%, many share structural similarities with non-viral proteins, suggesting functions like immune evasion. These findings provide new insights into virus-host interactions!

• Not yet a subscriber to Plenty of Room? Sign up today — it’s free!

• You think a friend or a colleague might enjoy reading this? Don’t hesitate to share it with them!

• Have a tip or story idea you want to share? Email me — I’d love to hear from you!

• You have something you would love me to cover? Just reach out here or on my social!