Press Release: Chemists Identify Key Step in Biosynthesis of Carbapenem Antibiotics
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PITTSBURGH—A team of researchers, including Carnegie Mellon University's Yisong Guo, have revealed a key step in the biosynthesis of carbapenems, a class of antibiotics used to treat some of the most serious drug-resistant bacterial infections. Their results were published in the March 7 issue of Science (doi: 10.1126/science.1248000). Understanding the chemical pathways that underlie the natural production of these molecules could help researchers develop new variants of antibiotics.
Carbapenem antibiotics are highly insusceptible to the bacterial enzymes that can cause drug resistance in other types of antibiotics, such as penicillins. The key to carbapenem's ability to avoid resistance is found in its unique chemical structure. Carbapenems contain beta-lactam/pyrrolidine rings that are almost inpenetrable to beta-lactamase enzymes produced by drug-resistant bacteria. This impenetrability comes courtesy of a stereo configuration at the end of the rings that is also key to conferring antibiotic properties on carbapenem molecules.
Researchers had found that early in the synthesis of carbapenem, the beta-lactam/pyrrolidine rings have an opposite stereo configuration at the end of the rings, which means they exist in a formation that is a mirror image of the final configuration. But how the final structure specifically evolved during chemical synthesis eluded scientists.
"We want to understand underlying chemical mechanisms for biosynthesis of natural products, in this case an antibiotic, through revealing key enzymatic intermediates in the biosynthetic pathways," said Guo, an assistant professor of chemistry in Carnegie Mellon's Mellon College of Science. "When molecules are synthesized by enzymes, chemical transformations usually happen quickly — each structural component may only exist for mere milliseconds. Stopping the biosynthetic pathway in order to study these transient events has proven to be challenging."
Using protein crystallography and advanced spectroscopic methods, coupled with transient enzyme kinetics and the rapid freeze quench technique, the researchers were able to capture and study key enzymatic intermediates during molecular biosynthesis.
In their study, Wei-chen Chang, a post-doctoral student at Penn State University, heterogeneously expressed CarC enzymes in E. coli and chemically synthesized carbapenem precursors, allowing for the in vitro study of the CarC enzyme. Using a rapid quenching technique honed by the Penn State group, which was led by Chemistry Professors J. Martin Bollinger, Jr., and Carsten Krebs, they stopped the production of the antibiotic molecule at critical steps during the production sequence. Guo, then a post-doctoral researcher at Penn State, used electron paramagnetic resonance (EPR) and Mössbauer spectroscopy to identify the chemical structures present at the different stages of synthesis.
After they analyzed the spectroscopic results, the research team was able to uncover the mechanism responsible for the stereo-inversion of the carbon configuration on the beta-lactam ring. They revealed that a key intermediate called Fe(IV)-oxo, removed a hydrogen atom of the carbon from one side of the molecule and then a tyrosine residue would donate a hydrogen to the opposite side to convert the molecule into its mirror image, thus completing the stereoinversion. This residue, tyrosine 165, had previously never been seen in CarC. Furthermore, this result also expands the chemical repertoire of Fe/alpha-KG dependent enzyme family to include a redox neutral stereoinversion reaction.
"You can't see these intermediates in the x-ray structure of the molecule, we needed more advanced methods to nail down the pathway," said Guo. "For the first time we have a clear result that shows how stereoinversion occurs in carbapenem biosynthesis."
In the future, the results could be used to create new drugs targeted at treating antibiotic resistant bacteria. Guo plans to apply the experimental methodology utilized in this study to understand the synthesis of other complex molecules in nature.
In addition to Guo, co-authors of the study include Penn State's Chang, Chen Wang, Susan E. Butch, Amie K. Boal, Krebs and Bollinger, and Northwestern University's Amy C. Rosenzweig. The research was supported by grants from the National Institutes of Health (GM 058518, GM 100011, and GM 069657).
A team of researchers, including CMU's Yisong Guo (pictured above), have revealed a key step in the biosynthesis of carbapenems, a class of antibiotics used to treat some of the most serious drug-resistant bacterial infections. Their work could help researchers develop new variants of antibiotics.