Real time molecular movie unlocks secrets of penicillin biosynthesis

Researchers from the University of Oxford and international collaborators have revealed previously unseen rapid chemical stages in the formation of β-lactam antibiotics like penicillin, offering insights that can support future antibiotic development. The findings have been published today (9 Oct) in Nature Catalysis.

Ever since penicillin was developed into a working drug at the University of Oxford in the early 1940's, β-lactam antibiotics have been among the most important medicines for treating bacterial infection. Their activity depends on a special ring - the β-lactam ring - a highly strained chemical ring system that interferes with bacterial cell wall synthesis, ultimately causing the cell wall to fail and the bacteria to die.

Scientists have been studying how nature constructs this β-lactam ring for decades, but the key fleeting reaction intermediates have been too difficult to observe directly.

Rising rates of antimicrobial resistance- a process in which bacteria, fungi and other microorganisms evolve to survive the medicines designed to kill them, are undermining the effectiveness of existing antibiotics. With too few new antibiotics in development, understanding how nature builds these molecules is an important step towards replenishing the antibiotic pipeline.

In the new study, researchers from the University of Oxford in collaboration with partners from Diamond Light Source, Lawrence Berkeley National Laboratory, PAL-XFEL and SLAC National Accelerator Laboratory have used X-ray free-electron lasers to observe the enzyme isopenicillin N synthase, or IPNS as it converts its linear peptide substrate into the ring system of penicillin.

The new study shows, in unprecedented detail, how the enzyme IPNS achieves an exceptionally complex transformation in a single step. This resolves a long-standing mechanistic question that has remained unanswered for more than four decades.

Rather than relying on static X-ray crystallographic structures of the enzyme, the researchers followed the reaction in real time using ultrafast X-ray free-electron laser (XFEL) experiments.

The team captured several rapid stages in the reaction. These included a thioaldehyde intermediate formed just before the β-lactam ring is created, and a monocyclic β-lactam intermediate, representing the first ring-shaped structure on the way to forming the complete penicillin scaffold. These steps provide the clearest picture yet of how the IPNS enzyme assembles the penicillin scaffold.

The work also showed that water molecules inside the enzyme play a key role in guiding the reaction. Subtle movements throughout the enzyme help guide these chemical steps, showing that both the enzyme's shape and its chemistry work together to control penicillin formation.

To capture the reaction in real time, the researchers used a system in which thousands of tiny droplets containing anaerobic enzyme microcrystals were deposited onto a moving 2 mm wide tape. As the tape entered an oxygen filled chamber, oxygen rapidly diffused into the crystals and initiated the reaction simultaneously across the sample. By precisely controlling the speed of the tape, the researchers could determine how long each crystal reacted before reaching the X-ray interaction point, where an ultrafast XFEL pulse recorded an atomic resolution snapshot.

Combining thousands of these snapshots allowed the team to build a frame-by-frame "molecular movie" of penicillin biosynthesis. This method allows individual reaction intermediates that exist only for tiny fractions of a second to be observed at atomic resolution, and under physiological temperature and pressure, before they disappear.

Oxford has played a central role in the history of penicillin since the pioneering work of Nobel prize-winning scientists Prof Howard Florey and Sir Ernst Chain, and colleagues, who developed penicillin into the first clinically useful antibiotic during the Second World War. This new study ultimately builds on the work of Nobel prize-winning Oxford chemist Dorothy Hodgkin, who first solved the structure of penicillin in 1945 using X-ray crystallography.

Beyond penicillin, IPNS belongs to a large family of iron-dependent oxygenase enzymes involved in human biology, including ones that enable us to sense and respond to changes in oxygen availability. The mechanistic insights from this work therefore have implications far beyond antibiotics, offering new principles for enzyme engineering and catalyst design.

Professor Christopher Schofield, Professor of Chemistry, University of Oxford, and a senior author of the paper, said, "Penicillin has shaped modern medicine, but there is still much to learn about how nature builds this important antibiotic structure. By capturing these fleeting steps, we can better understand how enzymes control complex chemistry with remarkable precision.

"As rates of antimicrobial resistance continue to rise, understanding this process will ultimately help us make existing antibiotics more efficient and design new antibiotic structures."

Today, one in six bacterial infections is resistant to antibiotics. Rising resistance threatens decades of progress in cancer care, transplant surgery and other areas of modern medicine, while also placing pressure on economies, health systems and food production.

These insights matter because they can inform how we think about antibiotic biosynthesis and future strategies for developing antibacterial medicines. The ability to capture structural snapshots over milliseconds to seconds of reaction time allows us to connect enzyme motion, iron chemistry and water-mediated proton transfer. This gives us a much richer view of how IPNS controls a difficult chemical transformation. By understanding this process in atomic detail, we can begin to think about engineering these enzymes to produce new or improved antibiotic scaffolds."

Dr. Patrick Rabe, Wellcome Career Development Award Investigator, University of Oxford and first author on the paper 

This study brought together expertise in structural biology, spectroscopy, chemistry, computation and enzyme engineering. This approach provides a powerful way to study fleeting chemical intermediates that have previously been inaccessible, both in antibiotic biosynthesis and in other biologically important enzyme reactions.

Dr Allen M Orville, group leader of the XFEL Hub and co-author of the paper, said: "The XFEL Hub at Diamond brought specialist expertise in time-resolved X-ray methods and XFEL-based structural biology to this study. Working closely with our colleagues in Oxford and at partner facilities, we helped bring together the experimental approaches needed to capture these fleeting stages of enzyme catalysis. The results show the power of combining complementary expertise and X-ray techniques to move beyond static structures and reveal how enzymes work in real time. It is a great example of how time-resolved structural biology can uncover new principles of enzyme function and, ultimately, inform the design of new catalysts and therapeutics."