Chemistry professor studies antibiotic-resistant bacteria

As the understanding of medical care has evolved, the use of antibiotics — specifically, the issue of antibiotic resistance — has become a major topic of discussion.

Yisong Guo, an assistant professor of chemistry at Carnegie Mellon, has become part of this conversation through his research regarding the biosynthesis of carbapenems, a class of antibiotics currently used to treat various types of drug-resistant bacterial infections.

A bacteria generally becomes resistant to antibiotics when it produces enzymes, such as beta-lactamase, which destroy antibiotic molecules and render the bacteria resistant. Fortunately, carbapenem antibiotics are insusceptible to these enzymes due to two unique structural components.

“The stereochemistry of the two protons on C5 and C6 carbons and the carbon-carbon double bonds at C2 and C3 carbons ... give carbapenem molecules protection against the attack of beta-lactamase,” Guo said. In other words, the unique spatial arrangement of atoms in carbapenems act as a defense against antibiotic resistance-inducing enzymes.

Researchers hypothesize that the formation of these two structural components is catalyzed by carbapenem synthase (CarC), a key enzyme in the synthesis of carbapenem that belongs to a class of enzymes called alpha-ketoglutarate (aKG) dependent mononuclear non-heme (MNH) iron enzymes. CarC is thought to initiate both the stereoinversion — a flip in structural orientation — that results in the two protons on C5 and C6 carbons, and the desaturation reaction that causes the carbon double bonds at C2 and C3.

The stereochemistry be- hind the C5 and C6 protons formed the basis for Guo’s re- search. It was previously dis- covered that precursors in the synthesis of carbapenem have the opposite stereochemistry from the final carbapenem on the two protons of C5 and C6 carbons, suggesting that carbapenem must go through a stereoinversion reaction in order to end up with its final structure.

Guo conducted the research while a postdoctoral researcher at Pennsylvania State University along with Wei-chen Chang, Chen Wang, Susan Butch, Amie Boal, Carsten Krebs, Amy Rosenzweig, and J. Martin Bollinger Jr.

The team’s research, which was supported by grants from the National Institutes of Health, aimed to determine the chemical mechanism behind the stereoinversion.

Guo explained that under- standing the chemical mechanism behind the carbapenem stereoinversion is essential for understanding many key biological processes. Uncovering this mechanism, however, produced many challenges. “Enzyme reactions are generally very fast, spanning the time scale from milliseconds to seconds, which renders traditional crystallographic technique not applicable,” Guo said. “One way to tackle this challenge is to use [a] rapid freeze-quench technique to stop the reaction at any given time point by quickly freezing the reaction solution, then to analyze the chemical species present in this frozen solution using spectroscopic techniques.”

Guo, a trained spectroscopist, used electron paramagnetic resonance (EPR) and Mössbauer spectroscopy to detect the structural components present during various levels of synthesis. Ultimately, the team was able to deter- mine the mechanism behind the stereoinversion. They determined that the molecule is situated between an iron center and an amino acid residue called tyrosine. An intermediate of carbapenem called Fe(IV)-oxo removes a hydrogen atom from one side of the molecule, after which the tyrosine donates a hydrogen atom to the opposite side of the molecule, completing the stereoinversion.

The discovery of this mechanism has the potential to impact many aspects of the chemical world. Guo explained that the research helps elucidate how enzymes such as carbapenem synthase work.

“It sheds light on how aKG- dependent MNH-Fe enzymes control their chemical reactivities through the protein scaffold surrounding their iron centers,” Guo said. “More importantly, it provides more knowledge to help us under- stand carbapenem antibiotics and create new drugs targeted at treating antibiotic resistant bacteria.”

Despite these results, Guo admits that there is still room for further study. “It is still unclear about the reaction mechanism on the desaturation reaction to form the C-C double bond,” Guo said. “It is likely that the enzyme starts new reaction cycles to build up the Fe(IV)-oxo intermediate ... but new experiment designs are needed to test this hypothesis.”

The discovery of new chemical mechanisms, such as the mechanism behind carbapenem stereoinversion, has the potential to greatly enhance our current knowledge of various biological and chemical processes.

This research paves the way for new, more effective antibiotics and could help determine the next steps in the prevention of antibiotic resistance.