Drug-resistant, Gram-negative bacteria are becoming more dangerous, causing a growing number of deaths worldwide. A newer class of natural antibiotics, darobactins, show significant promise in fighting these pathogens, but scientists haven’t fully understood how nature makes them.
Now a Duke University School of Medicine research team has revealed a long-sought missing step in how darobactins are produced in nature, pointing the way toward discovering or designing new, more effective antibiotics.
“We uncovered an elegant way nature builds powerful antibiotic molecules,” said graduate student Bach Nguyen, first author of the study, published in the Journal of the American Chemical Society. “Our work reveals how an enzyme, DarE, uses a short-lived chemical entity, known as a radical, to form an unprecedented carbon-oxygen bond that gives the antibiotics their strength and stability.”
In studying DarE, the researchers had previously found evidence that it uses molecular oxygen. They suspected that it did so by using a highly reactive radical. But they hadn’t caught it in the act.
In this study, using an advanced technique called electron paramagnetic resonance spectroscopy, the research team “trapped” the short-lived radical intermediate created by DarE during the reaction.
“DarE directly combines oxygen and the substrate, which is a rare strategy among all known enzymes,” said Ken Yokoyama, PhD, associate professor of biochemistry and senior author of the study. “The results provide insights into the mechanism by which enzymes use free radicals to incorporate oxygen into an organic substrate during the important antibiotic biosynthesis.”
Characterizing the structure and behavior of this radical overturns long-standing assumptions about how this class of enzymes operates and demonstrates that nature can safely harness oxygen in unexpected ways, he explained.
The R. David Britt lab at the University of California, Davis, and the Alex Smirnov lab at North Carolina State University provided their expertise in characterizing the radical using electron paramagnetic resonance spectroscopy.
As antibiotic resistance continues to rise, insights like these will be crucial for developing therapies that can stay ahead of dangerous pathogens.
Other Duke authors: Hai Nguyen, PhD, former postdoctoral associate and currently an assistant professor of chemistry at South Dakota School of Mines and Technology.
Funding: The National Institutes of Health, the American Heart Association, the National Science Foundation, the North Carolina Biotechnology Center.