CMU Chemists Describe How Non-Heme Iron Enzymes Form Aziridine from Free-Standing Amino Acids

A team of chemists from Carnegie Mellon University and North Carolina State University have identified two enzymes that can catalyze the stereo-specific formation of aziridine, a compound found in many natural products with potent antibiotic and antitumor activities. While nature has perfected aziridine formation, synthesizing aziridine and using it as a key intermediate to power chemical reactions is still challenging.

In the new work, published in the Journal of the American Chemical Society, the researchers determined on a molecular-level how two newly discovered iron-containing enzymes form aziridine from free-standing amino acids, a step toward repurposing these types of enzymes to perform larger-scale synthesis.

"You can find a lot of these types of enzymes in nature. They do incredible chemistry that synthetic chemists can only dream of," said Associate Professor of Chemistry Yisong (Alex) Guo. "Our study is fundamental, but the bigger implication is that if we can figure out how these enzymes function and how the enzymes' structure modulates their chemical reactivity, we can use that knowledge to develop novel synthetic methodology for natural product synthesis for antibiotic development and anti-cancer research."

Aziridines-three-membered rings made up of two carbons and a nitrogen-are found in several natural products isolated mainly from bacteria and fungi. The highly reactive ring drives aziridine's biological activity, making aziridine an attractive target for medicinal chemists looking to develop pharmaceuticals. To gain better insight into how nature forms aziridine, Guo and his colleague Wei-chen Chang, associate professor of chemistry at North Carolina State University, turned to fungi for inspiration.

The fungus Penicillium aethiopicum is known to contain a nonheme iron-containing enzyme (paTqaL) that catalyzes a chemical reaction to create aziridine from a free-standing amino acid, valine, ultimately producing a natural product with antibiotic properties. Chang took paTqaL's gene sequence and used sequence similarity network and genome-neighborhood network analysis to search through a gene database to identify additional enzymes with potential aziridine-formation ability. The research team found several candidates but chose to focus on two enzymes: one from the fungus Hypocrea atroviridis (haTqaL) and one from the fungus Penicillium digitatum (pdTqaL).

After expressing the two new enzymes in E. Coli and purifying them, Chang and his team used enzyme reaction product analysis to confirm that the enzymes generate aziridine from valine. Chang then fed the enzymes with several different amino acid substrates, and found that, depending on the substrate provided, the enzymes generated aziridine with different stereo-specificity. Interestingly, the type of substrate used could even alter the reactivity so that the enzyme carried out hydroxylation instead of aziridination.

Guo, along with graduate student Jared Paris and undergraduate student Martha Spletzer, dug deeper into the reaction mechanism. They carried out transient kinetics studies and spectroscopic characterizations to reveal the specific carbon site on the substrate where the initial oxidative activation occurs. They also discovered the key iron-based reactive intermediate responsible for aziridine and hydroxylation product formation.

The researchers used artificial-intelligence-based structure prediction software to predict the structure of the newly discovered enzymes. The AI-predicted structures are very close to the known crystal structure of the original TqaL enzyme. For Guo, this finding was particularly interesting.

"These enzymes structurally overlap very well. They are almost identical in terms of structure," Guo said. "So why do they show different reactivities when reacted with different substrates? How do they produce different products?"

Further analysis revealed that the key may be the enzymes' iron centers, where the chemical reactions take place. The substrate, the small molecule onto which the aziridine ring gets installed, is positioned at the iron center in a specific binding pose during the reaction. Guo's hypothesis is that the amino acids that surround the iron center are slightly different in each enzyme, which affects how the substrate is positioned, ultimately changing the trajectory of the reaction.

Guo is interested in further exploring the substrate binding platform to better understand which configuration does and doesn't promote aziridine formation. "That's the next level that we want to understand: What is the key structural factor that could enable aziridine formation in these group of enzymes."

Funding for the Carnegie Mellon portion of the study was provided by a grant from the National Institutes of Health and Carnegie Mellon's Summer Undergraduate Research Fellowship.