Bacteria and fungi have been engaged in molecular warfare for millions of years. This means they have perfected ways to get past the defenses of other organisms and have also devised ways to keep them out. This arms race was revealed in 1928 when Alexander Fleming returned from his holidays to discover a petri dish of bacteria in which a fungus had started to grow and was killing the bacteria around it. He immediately realized the potential value of these antibiotic molecules to humans for curing disease.
Now, however, our widespread use of natural antibiotics has led to the emergence of drug-resistant bacteria and an urgent need to develop some new molecular weapons of our own. With that in mind, a research group from the University of Michigan conducted a substrate-trapping study of bacterial enzymes that make an important class of antibiotics. The research provides important new information that will facilitate the design of new enzymes to make novel antibiotics that can overcome antibiotic resistance.
The group used the resources of the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) at beamlines 23-ID-B and 23-ID-D at the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.
The research focused on bacterial thioesterase (TE) enzymes that perform a critical step in a synthetic pathway to make macrolide antibiotics such as erythromycin and pikromycin. These TE enzymes temporarily attach antibiotic precursors to a nucleophilic amino acid in the TE, check the structural integrity of the precursor substrates, and then convert them to either a) a cyclic lactone molecule via nucleophilic attack by an oxygen atom in the substrate, or to b) a linear final product via attack by a water molecule. Although the structures of five TE enzymes that generate various products have been solved, the process by which a product is cyclized or hydrolyzed is poorly understood.
To get a clearer picture of the final step in the antibiotic synthesis process that might help researchers to understand the parameters needed to make new antibiotics, the team decided to use a technique called substrate trapping to visualize the moment of decision between cyclization and hydrolysis in different TE enzymes. They used a new substrate trapping technique that incorporates a non-natural amino acid into the active site in place of the natural serine or cysteine nucleophile. The bond attaching a substrate to serine or cysteine is unstable, but the non-natural amino acid traps the reaction intermediate as a stable amide group (see Figure).
After testing five bacterial TE enzymes to see if they could successfully incorporate the substrate trap, two of substrate trapping proteins could be purified in sufficient amounts for further testing, one that makes erythromycin and one that makes pikromycin, both cyclic antibiotics.
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Image: Model of the thioesterase enzyme active site with the cyclic substrate (purple) snugly fitted into the catalytic site of the TE (yellow). The substrate trap is represented by the blue nitrogen atom that forms a stable bond between the enzyme and substrate, preventing the substrate from leaving the site so the reaction intermediate can be studied at the molecular level. The substrate nucleophilic oxygen atom (red) is at the left end of the substrate.
Credit: Rajani Arora and Vishakha Choudhary of the University of Michigan.