How scientists are using unnatural amino acids to understand macrolactone formation and unlock new possibilities for medicine
Imagine a master craftsman who can twist a simple string into an intricate, life-saving bracelet. In the microscopic world of cells, this craftsman is an enzyme called a thioesterase, and the bracelets it forges are molecules known as macrolactones. These ring-shaped structures are the backbone of some of our most potent antibiotics, anti-cancer drugs, and immunosuppressants.
But how does this molecular craftsman know exactly when and how to twist the string into a perfect ring? Scientists are now using a brilliant trick—incorporating an unnatural amino acid as a molecular spy—to uncover these secrets .
This research reveals that the thioesterase domain is more flexible than previously thought, opening doors to engineering new drugs by reprogramming nature's own factories.
At their core, macrolactones are large rings, typically formed by 14, 15, or 16 atoms. This isn't just a quirky shape; it's a key to their function. Their structure allows them to interact precisely with the machinery of bacterial cells or cancer cells, disrupting their function without harming human cells .
Think of it like a key fitting into a lock. The complex, ring-shaped "key" (the macrolactone drug) fits perfectly into a specific "lock" (a target site on a ribosome, for instance) in a harmful bacterium, stopping it in its tracks. Famous drugs like Erythromycin (an antibiotic) and Rapamycin (an immunosuppressant) are all macrolactones, produced naturally by bacteria and fungi.
A widely used antibiotic effective against various bacterial infections, particularly respiratory tract infections.
An immunosuppressant used in organ transplantation and studied for its potential anti-aging properties.
The big question has always been: how do the producer organisms build these rings with such perfect size and consistency?
The answer lies in massive molecular assembly lines called Non-Ribosomal Peptide Synthetases (NRPS) and related systems for polyketides. These are like factory conveyor belts, where each station (or domain) adds a new piece to a growing molecular chain.
Simplified representation of the NRPS assembly line with the thioesterase domain as the final step
At the very end of this assembly line sits the crucial final worker: the Thioesterase (TE) Domain. Its job is to recognize when the chain is long enough and then perform the delicate operation of cutting it from the assembly line and coaxing it into a ring. This process is called cyclization or macrolactonization.
For decades, scientists have been trying to understand the TE domain's rulebook. Does it simply count the number of units in the chain? Does it feel for a specific 3D shape? The new research using an unnatural amino acid has provided some of the clearest answers yet .
To understand the TE domain's preferences, a team of scientists devised an ingenious plan. Instead of just watching the natural process, they decided to feed the assembly line a building block that shouldn't normally be there—a molecular spy that would report back on the TE domain's true flexibility.
Researchers selected an unnatural amino acid called L-Homopropargylglycine (HPG). This molecule is similar enough to a natural amino acid (methionine) that the cellular machinery would mistakenly incorporate it into the growing chain. Its key feature is a "clickable" alkyne group—a chemical handle that acts like a tiny hook .
They used genetic engineering to create a model bacterial system whose NRPS assembly line would be tricked into using HPG at a specific position in the chain, right before the cyclization point.
They fed this engineered bacteria a diet containing HPG instead of its usual methionine. The bacteria obediently started production, incorporating the spy molecule into the growing chain.
The chain, now containing HPG, eventually reached the TE domain. The critical moment: would the TE domain still recognize this altered chain as a valid substrate and perform the cyclization to form the macrolactone ring?
After giving the bacteria time to produce the molecules, the team harvested the culture. They then used a special "click chemistry" reaction—exploiting the unique hook on the HPG spy—to fish out and isolate only the macrolactone products that contained the unnatural amino acid .
Creating bacterial factories programmed to incorporate unnatural amino acids
Using specialized reactions to isolate and identify novel macrolactones
The results were striking. The TE domain did, in fact, successfully cyclize the chains containing HPG, producing novel macrolactones that do not exist in nature.
This discovery opens the door to biosynthetic engineering, a field aimed at designing new drugs by reprogramming nature's own factories to produce novel compounds with potential therapeutic applications.
| Precursor Chain Length | Natural Amino Acid (Control) | HPG Incorporated | Cyclization Efficiency (Relative %) |
|---|---|---|---|
| 12 atoms | Yes | Yes | 85% |
| 14 atoms | Yes | Yes | 95% |
| 16 atoms | Yes | Yes | 78% |
| Product ID | Chain Length | Contains HPG? | Ring Size Formed | Relative Abundance |
|---|---|---|---|---|
| ML-A | 14 atoms | No | 14-membered | 100% |
| ML-B | 14 atoms | Yes | 14-membered | 92% |
| ML-C | 16 atoms | Yes | 16-membered | 45% |
The simple act of incorporating an unnatural amino acid has given us an unprecedented look into the workshop of a molecular craftsman. By revealing the flexibility of the thioesterase domain, this research does more than just satisfy scientific curiosity—it provides a powerful new blueprint .
Combat antibiotic resistance by designing novel macrolactones effective against superbugs
Engineer macrolactones that specifically target cancer cells with fewer side effects
Reprogram natural pathways to produce entirely new classes of therapeutic compounds
We are no longer limited to the drugs that nature has already made. By understanding and hacking these biosynthetic pathways, scientists can now design and produce a new generation of "designer" macrolactones, potentially leading to more effective antibiotics to fight superbugs, targeted cancer therapies with fewer side effects, and a new arsenal of medicines for the challenges of tomorrow. The molecular LEGO kit is now in our hands.